Low affinity blood brain barrier receptor antibodies and uses therefor

ABSTRACT

The present invention relates to antibodies that bind blood brain barrier receptors (BBB-R) and methods of using the same.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional application No. 61/418,223, filed Nov. 30, 2010, thecontents of which are incorporated in their entirety herein byreference.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind blood brainbarrier receptors (BBB-R) and methods of using the same.

BACKGROUND

Brain penetration of large molecule drugs is severely limited by thelargely impermeable blood-brain barrier (BBB). Among the many strategiesto overcome this obstacle is to utilize transcytosis traffickingpathways of endogenous receptors expressed at the brain capillaryendothelium. Recombinant proteins such as monoclonal antibodies havebeen designed against these receptors to enable receptor-mediateddelivery of large molecules to the brain. However, strategies tomaximize brain uptake while minimizing reverse transcytosis back to theblood, and the extent of accumulation after therapeutic dosing, remainunexplored. Furthermore, whether antibodies that cross the BBB arepharmacodynamically functional is unknown.

SUMMARY

Monoclonal antibodies have vast therapeutic potential for treatment ofneurological or central nervous system (CNS) diseases, but their passageinto the brain is restricted by the blood-brain barrier (BBB). Paststudies have shown that a very small percentage (approximately 0.1%) ofan IgG circulating in the bloodstream crosses through the BBB into theCNS (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)), where the CNSconcentration of the antibody may be insufficient to permit a robusteffect. The methods and compositions of the invention provide a way toimprove the percentage of the antibody that distributes into the CNS andthus more readily attain therapeutic antibody concentrations in the CNS.

Herein is described a group of antibodies against the transferrinreceptor (TfR) that can deliver therapeutics including antibodies andsmall molecules across the BBB at both trace and therapeuticallyrelevant doses after a single systemic injection in mice. Distributionof antibody changed from vascular to neuronal 24 hours after injection,indicating that a significant amount of antibody had transcytosedthrough brain endothelial cells to reach the parenchyma. The magnitudeof antibody uptake into and distribution in the CNS was inverselyrelated to its binding affinity to TfR for the anti-TfR variantsstudied. Proof of BBB transport was achieved using a bispecific antibodythat binds both TfR and the amyloid precursor protein (APP) cleavageenzyme, β-secretase (BACE1). A single systemic dose of the bispecificanti-TfR/BACE1 antibody engineered using the methodology of theinvention not only resulted in significant antibody uptake in brain, butalso dramatically reduced levels of brain Aβ₁₋₄₀ compared tomonospecific anti-BACE1 alone, suggesting that BBB penetrance affectsthe potency of anti-BACE1. Similarly, a bispecific antibody that bindsboth TfR and amyloid beta (i.e., a portion of APP that results fromBACE1 cleavage of APP, which is one of the main constituents of amyloidplaques) was shown to be readily taken up into the brain using themethodology of the invention. The data and experiments described hereinhighlight several causative mechanisms behind increasing uptake of anantibody into the CNS using a lower-affinity antibody approach. First,high affinity anti-BBB receptor (BBB-R) antibodies (e.g., anti-TfR^(A))limit brain uptake by quickly saturating the BBB-R in the brainvasculature, thus reducing the total amount of antibody taken up intothe brain and also restricting its distribution to the vasculature.Strikingly, lowering affinity for the BBB-R improves brain uptake anddistribution, with a robust shift observed in localization from thevasculature to neurons and associated neuropil distributed within theCNS. Second, the lower affinity of the antibody for the BBB-R isproposed to impair the ability of the antibody to return to the vascularside of the BBB via the BBB-R from the CNS side of the membrane becausethe overall affinity of the antibody for the BBB-R is low and the localconcentration of the antibody on the CNS side of the BBB isnon-saturating due to the rapid dispersal of the antibody into the CNScompartment. Third, in vivo, and as observed for the TfR system,antibodies with less affinity for the BBB-R are not cleared from thesystem as efficiently as those with greater affinity for the BBB-R, andthus remain at higher circulating concentrations than theirhigher-affinity counterparts. This is advantageous because thecirculating antibody levels of the lower-affinity antibody are sustainedat therapeutic levels for a longer period of time than thehigher-affinity antibody, which consequently improves uptake of antibodyin brain for a longer period of time. Furthermore, this improvement inboth plasma and brain exposure may reduce the frequency of dosing in theclinic, which would have potential benefit not only for patientcompliance and convenience but also in lessening any potential sideeffects or off-target effects of the antibody and/or of a therapeuticcompound coupled thereto. Anti-TfR/BACE1 and anti-TfR/Abeta are eachpromising and novel therapeutic candidates for the treatment ofAlzheimer's disease. Furthermore, receptor mediated transport(RMT)-based bispecific targeting technology opens the door for a Widerange of potential therapeutics for CNS diseases. The invention providesmethods of engineering BBB-penetrant therapeutics that greatly improvetransport across the BBB and CNS distribution of the therapeutic.

Accordingly, in a first embodiment, the invention provides a method oftransporting a compound across the blood-brain barrier comprisingexposing an antibody which binds with low affinity to a blood-brainbarrier receptor (BBB-R) coupled to a compound to the blood-brainbarrier such that the antibody transports the compound coupled theretoacross the blood-brain barrier. In one aspect, the compound is aneurological disorder drug. In another aspect, the compound is animaging agent. In another aspect, the compound is labeled. In anotheraspect, the antibody is labeled. In another aspect, the antibody doesnot impair the binding of the BBB-R to one or more of its nativeligands. In another such aspect, the antibody specifically binds to TfRin such a manner that it does not inhibit binding of the TfR totransferrin. In another aspect, the BBB is in a mammal. In another suchaspect, the mammal is a human. In another such aspect, the mammal has aneurological disorder. In another such aspect, the neurological disorderis selected from the group consisting of Alzheimer's disease (AD),stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS),amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman'ssyndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget'sdisease, cancer, and traumatic brain injury. In another aspect, the BBBis in a human.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment, the invention provides a method of increasingexposure of the CNS to a compound, wherein the compound is coupled to anantibody which binds with low affinity to a BBB-R, thereby increasingthe exposure of the CNS to the compound. In one aspect, the compound isa neurological disorder drug. In another aspect, the compound is animaging agent. In another aspect, the compound is labeled. In anotheraspect, the antibody is labeled. In another aspect, the antibody doesnot impair the binding of the BBB-R to one or more of its nativeligands. In another such aspect, the antibody specifically binds to TfRin such a manner that it does not inhibit binding of the TfR totransferrin. In another aspect, the antibody-coupled compound isadministered to a mammal. In another such aspect, the mammal is a human.In another such aspect, the mammal has a neurological disorder. Inanother such aspect, the neurological disorder is selected from thegroup consisting of Alzheimer's disease (AD), stroke, dementia, musculardystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis(ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome,Parkinson's disease, Pick's disease, Paget's disease, cancer, andtraumatic brain injury.

In another aspect, the increase in CNS exposure to the compound ismeasured relative to the CNS exposure of a compound coupled with atypical antibody not having lowered affinity for the BBB-R. In anotheraspect, the increase in CNS exposure to the compound is measured as aratio of the amount of the compound found in the CNS relative to theamount found in the serum after administration. In another such aspect,the increase in CNS exposure results in a ratio of greater than 0.1%. Inanother aspect, the increase in CNS exposure to the compound is measuredrelative to the CNS exposure of a compound in the absence of a coupledantibody. In another aspect, the increase in CNS exposure to thecompound is measured by imaging. In another aspect, the increase in CNSexposure to the compound is measured by an indirect readout such as amodification of one or more physiological symptoms.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP 1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment, the invention provides a method of decreasingclearance of a compound administered to a subject, wherein the compoundis coupled to an antibody which binds with low affinity to a BBB-R, suchthat the clearance of the compound is decreased. In one aspect, thecompound is a neurological disorder drug. In another aspect, thecompound is an imaging agent. In another aspect, the compound islabeled. In another aspect, the antibody is labeled. In another aspect,the antibody does not impair the binding of the BBB-R to one or more ofits native ligands. In another such aspect, the antibody specificallybinds to TfR in such a manner that it does not inhibit binding of the URto transferrin. In another aspect, the subject is a mammal. In anothersuch aspect, the mammal is a human. In another such aspect, the mammalhas a neurological disorder. In another such aspect, the neurologicaldisorder is selected from the group consisting of Alzheimer's disease(AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis(MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman'ssyndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget'sdisease, cancer, and traumatic brain injury.

In another aspect, the decrease in clearance of the compound is measuredrelative to the clearance of a compound coupled with a typical antibodynot having lowered affinity for the BBB-R. In another aspect, thedecrease in clearance of the compound is measured relative to theclearance of the compound in the absence of a coupled antibody.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

A method of increasing retention in the CNS of a compound administeredto a subject, wherein the compound is coupled to an antibody which bindswith low affinity to a BBB-R, such that the retention in the CNS of thecompound is increased. In one aspect, the compound is a neurologicaldisorder drug. In another aspect, the compound is an imaging agent. Inanother aspect, the compound is labeled. In another aspect, the antibodyis labeled. In another aspect, the antibody does not impair the bindingof the BBB-R to one or more of its native ligands. In another suchaspect, the antibody specifically binds to TfR in such a manner that itdoes not inhibit binding of the TfR to transferrin. In another aspect,the compound is administered to a mammal. In another such aspect, themammal is a human. In another such aspect, the mammal has a neurologicaldisorder. In another such aspect, the neurological disorder is selectedfrom the group consisting of Alzheimer's disease (AD), stroke, dementia,muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateralsclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome,Parkinson's disease, Pick's disease, Paget's disease, cancer, andtraumatic brain injury.

In another aspect, the increase in CNS retention of the compound ismeasured relative to the CNS retention of a compound coupled with atypical antibody not having lowered affinity for the BBB-R. In anotheraspect, the increase in CNS retention of the compound is measured as aratio of the amount of the compound found in the CNS relative to theamount found in the serum at one or more time points afteradministration. In another such aspect, the increase in CNS retentionresults in a ratio of greater than 0.1% at one or more time points afteradministration. In another aspect, the increase in CNS retention of thecompound is measured relative to the CNS retention of a compound in theabsence of a coupled antibody. In another aspect, the increase in CNSretention of the compound is measured by imaging. In another aspect, theincrease in CNS retention of the compound is measured by an indirectreadout such as a modification of one or more physiological symptoms.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment, the invention provides a method of optimizing thepharmcokinetics and/or pharmacodynamics of a compound to be efficaciousin the CNS of a subject, wherein the compound is coupled to an antibodywhich binds with low affinity to a BBB-R, and the antibody is selectedsuch that its affinity for the BBB-R after coupling to the compoundresults in an amount of transport of the antibody conjugated to thecompound across the BBB that optimizes the pharmacokinetics and/orpharmacodynamics of the compound in the CNS. In one aspect, the compoundis a neurological disorder drug. In another aspect, the compound is animaging agent. In another aspect, the compound is labeled. In anotheraspect, the antibody is labeled. In another aspect, the antibody doesnot impair the binding of the BBB-R to one or more of its nativeligands. In another such aspect, the antibody specifically binds to TfRin such a manner that it does not inhibit binding of the TfR totransferrin. In another aspect, the BBB is in a mammal. In another suchaspect, the mammal is a human. In another such aspect, the mammal has aneurological disorder. In another such aspect, the neurological disorderis selected from the group consisting of Alzheimer's disease (AD),stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS),amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman'ssyndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget'sdisease, cancer, and traumatic brain injury. In another aspect, the BBBis in a human.

In one aspect, the optimizing may include the generation of a series ofantibody-compound complexes in which each antibody has a differentaffinity for the BBB-R, and assessing the pharmacokinetics and/orpharmacodynamics of each in the CNS. In another aspect, optimizing maybe relative to a known standard, such as, but not limited to, thepharmacokinetics and/or pharmacodynamics of the compound when directlyintroduced into the CNS or when introduced to the subject in the absenceof a coupled anti-BBB-R antibody.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment the invention provides a method of treating aneurological disorder in a mammal comprising treating the mammal with anantibody that binds a BBB-R and is coupled to a compound, wherein theantibody has been selected to have a low affinity for the BBB-R andthereby improves CNS uptake of the antibody and coupled compound. In oneaspect, the compound is a neurological disorder drug. In another aspect,the compound is an imaging agent. In another aspect, the compound islabeled. In another aspect, the antibody is labeled. In another aspect,the antibody does not impair the binding of the BBB-R to one or more ofits native ligands. In another such aspect, the antibody specificallybinds to TfR in such a manner that it does not inhibit binding of theTfR to transferrin. In one aspect, the mammal is a human. In anothersuch aspect, the mammal has a neurological disorder. In another suchaspect, the neurological disorder is selected from the group consistingof Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD),multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cysticfibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease,Pick's disease, Paget's disease, cancer, and traumatic brain injury.

In one aspect, the treating results in lessening or elimination ofdisorder symptoms. In another aspect, the treating results inamelioration of the neurological disorder.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 100 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment, the invention provides a method of making anantibody useful for transporting a compound across the BBB comprisingselecting an antibody specific for a blood-brain barrier receptor(BBB-R) because it has a desirably low affinity for the BBB-R.

In one aspect, the antibody is selected from a panel of antibodies basedupon the affinity of the selected antibody. In another aspect, theantibody is engineered to have the affinity. In one such aspect, theantibody is generated using any art-known protein engineeringmethodology including, but not limited to, phage display, yeast display,random mutagenesis, and site-directed mutagenesis.

In one aspect, the compound is a neurological disorder drug. In anotheraspect, the compound is an imaging agent. In another aspect, thecompound is labeled. In another aspect, the antibody is labeled. Inanother aspect, the antibody does not impair the binding of the BBB-R toone or more of its native ligands. In another such aspect, the antibodyspecifically binds to TfR in such a manner that it does not inhibitbinding of the TfR to transferrin. In another aspect, the BBB is in amammal. In another such aspect, the mammal is a human. In another suchaspect, the mammal has a neurological disorder. In another such aspect,the neurological disorder is selected from the group consisting ofAlzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD),multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cysticfibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease,Pick's disease, Paget's disease, cancer, and traumatic brain injury. Inanother aspect, the BBB is in a human.

In another aspect, the antibody has an IC50 for the BBB-R from about 1nM to about 100 μM. In another such aspect, the IC50 is from about 5 nMto about 100 μM. In another such aspect, the IC50 is from about 50 nM toabout 100 μM. In another such aspect, the IC50 is from about 100 nM toabout 100 μM. In another aspect, the antibody has an affinity for theBBB-R from about 5 nM to about 10 μM. In another such aspect, theantibody, when coupled to a compound, has an affinity for the BBB-R fromabout 30 nM to about 1 μM. In another such aspect, the antibody, whencoupled to a compound, has an affinity for the BBB-R from about 50 nM toabout 1 μM. In another such aspect, the compound-coupled antibodyspecifically binds to TfR and has an affinity for TfR between thoseaffinities observed for the anti-TfR^(A)/BACE1 antibody and theanti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(D)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has an IC50for TfR between those IC50s observed for the anti-TfR^(A)/BACE1 antibodyand the anti-TfR^(E)/BACE1 antibody. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an IC50 forTfR between those IC50s observed for the anti-TfR^(D)/BACE1 antibody andthe anti-TfR^(E)/BACE1 antibody. In one aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured usingscatchard analysis. In another aspect, the affinity of the anti-BBB-R oranti-BBB-R/compound for the BBB-R is measured using BIACORE analysis. Inanother aspect, the affinity of the anti-BBB-R or anti-BBB-R/compoundfor the BBB-R is measured using a competition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In another such aspect, the BBB-R is a human BBB-R. In onesuch aspect, the BBB-R is TfR. In another such aspect, the BBB-R is TfRand the antibody does not inhibit TfR activity. In another such aspect,the BBB-R is TfR and the antibody does not inhibit the binding of TfR totransferrin. In another aspect, the compound-coupled antibody isadministered at a therapeutic dose. In one such aspect, the therapeuticdose is a dose that saturates the BBB-R to which the antibodyspecifically binds.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another embodiment, the invention provides an antibody which binds toa blood-brain barrier receptor (BBB-R) with low affinity. In one aspect,the affinity of the antibody for the BBB-R is from about 5 nM to about10 μM. In another aspect, the affinity of the antibody for the BBB-R isfrom about 20 nM to about 1 μM. In another aspect, the antibody has anIC50 for the BBB-R from about 1 nM to about 100 μM. In another suchaspect, the IC50 is from about 5 nM to about 100 μM. In another suchaspect, the IC50 is from about 50 nM to about 100 μM. In another suchaspect, the IC50 is from about 100 nM to about 100 μM. In another suchaspect, the antibody, when coupled to a compound, has an affinity forthe BBB-R from about 50 nM to about 1 μM. In another such aspect, thecompound-coupled antibody specifically binds to TfR and has an affinityfor TfR between those affinities observed for the anti-TfR^(A)/BACE1antibody and the anti-TfR^(E)/BACE1 antibody. In another such aspect,the compound-coupled antibody specifically binds to TfR and has anaffinity for TfR between those affinities observed for theanti-TfR^(D)/BACE1 antibody and the anti-TfR^(E)/BACE1 antibody. Inanother such aspect, the compound-coupled antibody specifically binds toTfR and has an IC50 for TfR between those IC50s observed for theanti-TfR^(A)/BACE1 antibody and the anti-TfR^(E)/BACE1 antibody. Inanother such aspect, the compound-coupled antibody specifically binds toTfR and has an IC50 for TfR between those IC50s observed for theanti-TfR^(D)/BACE1 antibody and the anti-TfR^(E)/BACE1 antibody. In oneaspect, the affinity of the anti-BBB-R or anti-BBB-R/compound for theBBB-R is measured using scatchard analysis. In another aspect, theaffinity of the anti-BBB-R or anti-BBB-R/compound for the BBB-R ismeasured using BIACORE analysis. In another aspect, the affinity of theanti-BBB-R or anti-BBB-R/compound for the BBB-R is measured using acompetition ELISA.

In another aspect, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 8 (LRP8), low density lipoprotein receptor-related protein 1(LRP1), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). In one such aspect, the BBB-R is TfR. In another such aspect,the BBB-R is TfR and the antibody does not inhibit TfR activity. Inanother such aspect, the BBB-R is TfR and the antibody does not inhibitthe binding of TfR to transferrin. In another such aspect, the BBB-R isa human BBB-R.

In another aspect, the antibody is coupled to a compound. In one aspect,the compound is a neurological disorder drug. In another aspect, thecompound is an imaging agent. In another aspect, the compound islabeled. In another aspect, the antibody is labeled. In another aspect,the antibody does not impair the binding of the BBB-R to one or more ofits native ligands. In another such aspect, the antibody specificallybinds to TfR in such a manner that it does not inhibit binding of theTfR to transferrin.

In another aspect, the compound is covalently coupled to the antibody.In one such aspect, the compound is joined to the antibody by a linker.In one such aspect, the linker is cleavable. In another such aspect, thelinker is not cleavable. In another such aspect, the compound isdirectly linked to the antibody. In one such aspect, the antibody is amultispecific antibody and the compound forms one portion of themultispecific antibody. In another such aspect, the multispecificantibody comprises a first antigen binding site which binds the BBB-Rand a second antigen binding site which binds a brain antigen. Inanother such aspect, the brain antigen is selected from the groupconsisting of: beta-secretase 1 (BACE1), Abeta, epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In another such aspect, the multispecific antibody binds both TfR andBACE1. In another such aspect, the multispecific antibody binds both TfRand Abeta. In another such aspect, the multispecific antibody islabeled. In another aspect, the compound is reversibly coupled to theantibody such that the compound is released from the antibody concurrentwith or after BBB transport.

In another aspect, the antibody is an antibody fragment with anantigen-binding region that binds the BBB-R, including, but not limitedto, Fab, Fab′, F(ab′)₂, and Fv. In another aspect, the antibody is afull-length antibody.

In another embodiment, the invention provides the use of an antibodythat binds with low affinity to a BBB-R for the manufacture of amedicament for treating a neurological disorder. Any of the foregoingdescribed low-affinity anti-BBB-R antibodies or any of the low-affinityanti-BBB-R antibodies described elsewhere herein may be used in themethod.

In another embodiment, the invention provides an antibody that bindswith low affinity to a BBB-R for use in treating a neurologicaldisorder. Any of the foregoing described low-affinity anti-BBB-Rantibodies or any of the low-affinity anti-BBB-R antibodies describedelsewhere herein may be used in the method. Accordingly, in a firstaspect, the invention provides an antibody which binds to a blood-brainbarrier receptor (BBB-R), wherein the affinity of the antibody for theBBB-R is from about 5 nM to about 10 μM (e.g. from about 20 nM to about1 μM). Optionally, the BBB-R is selected from the group consisting oftransferrin receptor (TfR), insulin receptor, insulin-like growth factorreceptor (IGF receptor), low density lipoprotein receptor-relatedprotein 1 (LRP1), low density lipoprotein receptor-related protein 8(LRP8), and heparin-binding epidermal growth factor-like growth factor(HB-EGF). Optionally, the antibody is coupled with a therapeuticcompound, such as a neurological disorder drug. In one embodiment, theantibody is a multispecific antibody which comprises a first antigenbinding site which binds the BBB-R and a second antigen binding sitewhich binds a brain antigen, for instance where the brain antigen isselected from the group consisting of: beta-secretase 1 (BACE1), amyloidbeta (Abeta), epidermal growth factor receptor (EGFR), human epidermalgrowth factor receptor 2 (HER2), Tau, apolipoprotein A3 (ApoE3),apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, Huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. The antibody (e.g. multispecific antibody) includes antibodyfragments and full-length antibodies.

In another embodiment, the invention provides a method of transporting atherapeutic compound, such as a neurological disorder drug, across theblood-brain barrier comprising exposing the anti-BBB-R antibody coupledwith a neurological disorder drug to the blood-brain barrier such thatthe antibody transports the neurological disorder drug coupled theretoacross the blood-brain barrier. The blood-brain barrier in this methodmay be in a mammal, e.g. one with a neurological disorder, examples ofwhich include: Alzheimer's disease (AD) (including, but not limited to,mild cognitive impairment and prodromal AD), stroke, dementia, musculardystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis(ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome,Parkinson's disease, Pick's disease, Paget's disease, cancer (e.g.cancer affecting the CNS or brain), and traumatic brain injury.

The invention additionally concerns a method of making an antibodyuseful for transporting a therapeutic compound such as a neurologicaldisorder drug across the blood-brain barrier comprising selecting anantibody against a blood-brain barrier receptor (BBB-R) because it hasan affinity for the BBB-R which is from about 5 nM to about 10 μM. Inone embodiment the antibody is selected from a panel of antibodiesbecause it has the desired affinity. Alternatively, or additionally, theantibody is engineered to have the desired affinity. The methodoptionally further comprises coupling the antibody with a therapeuticcompound such as a neurological disorder drug. For example, the methodcan comprise making a multispecific antibody which comprises a firstantigen binding site which binds the BBB-R and a second antigen bindingsite which binds a brain antigen.

The invention additionally provides a method of treating a neurologicaldisorder in a mammal comprising treating the mammal with a multispecificantibody that binds both a blood-brain barrier receptor (BBB-R) and abrain antigen, wherein the anti-BBB-R antibody has been selected to havea low affinity for the BBB-R and thereby improves brain uptake of theanti-brain antigen antibody. Optionally, the multispecific antibodybinds both transferrin receptor (TfR) and BACE1 or Abeta.

It will be understood that any of the foregoing methods and compositionsof the invention may be combined with one another and/or with thefurther aspects of the invention described in the specification herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-E depict significant brain vascular uptake of systemicallyadministered anti-TfR antibody. FIG. 1A shows brain uptake after IVadministration of trace doses (approximately 50 μg/kg) of[¹³¹I]anti-TfR^(A) and [¹²⁵I]control IgG in mice and was quantified as amean percentage of injected dose per gram of brain at 5, 30 min., 1, 4,24, 48, and 72 hours after IV injection (n=6). Uptake of[¹³¹I]anti-TfR^(A) was decreased by injection with 4 mg/kg unlabeledanti-TfR^(A) (cold). FIG. 1B shows quantification of mean antibodyuptake in brain 1 and 24 hours after a 20 mg/kg IV injection of controlIgG or anti-TfR^(A) (***p=0.0002, n=10). FIG. 1C shows ratio of meanpercent brain to serum concentrations (**p=0.003, n=10). FIGS. 1D and 1Edepict immunohistochemical staining of brain sections followingIV-injection with anti-TfR^(A) (FIG. 1D, upper panels), which showsco-localization with anti-collagen IV, a vascular marker (lower panel).IV-injection with control IgG (FIG. 1E, upper panels) exhibits vasculardistribution in brain only after 1 hour and an absence of antibody after24 hours. Scale bar=50 μm.

FIGS. 2A-F show that affinity of anti-TfR antibodies and extent of brainuptake are inversely related when administered at a therapeuticallyrelevant dose (20 mg/kg) as compared to a low trace dose (approximately50 μg/kg). FIG. 2A depicts a competitive binding ELISA in whichincreasing concentrations of anti-TfR^(A,B,C,D,E) variant antibodies areused to compete against biotinylated TfR^(A) for binding to TfR. Theanti-TfR competition ELISA was performed in Maxisorp plates (Neptune,N.J.) coated with 2.5 μg/ml of purified murine TfR extracellular domainin PBS at 4° C. overnight. Plates were washed with PBS/0.05% Tween 20and blocked using Superblock blocking buffer in PBS (Thermo Scientific,Hudson, N.H.). A titration of anti-TfR^(A), anti-TfR^(B), anti-TfR^(C),or anti-TfR^(D) (1:3 serial dilution) was combined with biotinylatedanti-TfR^(A) (0.5 nM final concentration) and added to the plate for 1hour at room temperature. Plates were washed with PBS/0.05% Tween 20,and HRP-streptavidin (Southern Biotech, Birmingham) was added to theplate and incubated for 1 hour at room temperature. Plates were washedwith PBS/0.05% Tween 20, and biotinylated anti-TfR^(A) bound to theplate was detected using TMB substrate (BioFX Laboratories, OwingsMills). The results in FIG. 2A present data from a single experiment inwhich all five anti-TfR variants were separately assessed. The 1050values determined from this data are shown in Table 2. FIG. 2B depictsquantification of mean brain [¹²⁵I]anti-TfR^(A,B,C,D,E) uptake afterIV-injection of trace doses (approximately 50 μg/kg) of the variantsafter 5 min., 1, 4, 6, and 24 hours (n=3). The results in FIG. 2Bpresent data from a single experiment in which all five anti-TfRvariants were separately assessed. FIG. 2C shows quantification of meanbrain uptake after 20 mg/kg IV injection of anti-TfR variants at 1 and24 hours using the methods described with regard to FIG. 1B. Theexperiment was replicated under the same conditions using anti-TfR^(E),and all results presented in FIG. 2C. FIG. 2D is a model illustratingthe inverse relationship between affinity and brain uptake. FIG. 2E is acomparison of immunohistochemical staining of brain sections after IVinjection with either the high affinity anti-TfR^(A) or lower affinityanti-TfR^(B,C,D) antibodies showing differences in antibody distribution(staining in left panels is for anti-TfR alone) and extent ofco-localization with NeuN (staining in right panels is for both anti-TfRand NeuN). Scale bar=50 μm. FIG. 2F is a representative highmagnification image of anti-TfR^(D) localizaton in neurons (as indicatedby NeuN staining); this data shows that anti-TfR^(D) and NeuN colocalizeand thus that anti-TfR^(D) traverses the BBB and interacts with neurons,whereas anti-TfR^(A) mainly localizes to the vasculature as opposed toneurons. Scale bar=20 μm.

FIGS. 3A-G show that a bispecific anti-TfR/BACE1 antibody inhibits Aβ invitro and accumulates in the brain. FIG. 3A is a schematic model of abispecific antibody which was engineered to bind both TfR andβ-secretase (BACE1). FIG. 3B shows binding affinity for TfR of theparental anti-TfR^(A) and anti-TfR^(A)/BACE1 as measured by the anti-TfRcompetition ELISA assay described above for FIG. 2A. FIG. 3C showsquantification of Aβ levels produced by HEK293 cells stably expressingAPP after treatment with anti-TfR^(A)/BACE1, anti-BACE1, and control IgGin a cell-based assay. The ability of the antibodies to inhibit Aβ1-40production in HEK293 cells stably expressing wild-type human amyloidprecursor protein was assessed as follows. HEK293-APPWT cells wereseeded overnight at a density of 3×10⁴ cells/well in a 96-well plate. 50μl of fresh media (DMEM+10% FBS) containing an anti-BACE1 antibody or acontrol IgG1 antibody was incubated with the cells for 24 hours at 37°C. The cellular media was harvested and assayed for the presence ofAβ1-40 using a Aβ1-40 HTRF® assay (CisBio) according to themanufacturer's instructions. Aβ1-40 values were normalized for cellviability, as determined using the CellTiter-Glo Luminescent CellViability Assay (Promega). Experiments were performed at least threetimes, and each point in each experiment was repeated in duplicate. FIG.3D depicts quantification of mean brain uptake after trace doses of[¹²⁵I]-labeled antibody 30 min., 6, 24, and 48 hours after IV-injectionin mice (n=4). FIG. 3E shows quantification of mean antibody uptake inbrain and in FIG. 3F average brain to serum ratio at 1, 12, 24, and 48hours after a 20 mg/kg IV injection of antibody in mice (n=10). Theexperiments in FIGS. 3E and 3F was performed using the same protocol asthe experiment described with regard to FIG. 1B. FIG. 3G showsimmunohistochemical staining of brain sections from mice 24 hours afterIV injection with either anti-TfR/BACE1 (left panels) or control IgG(right panels). Co-localization of antibody with NeuN is observed afteranti-TfR^(A)/BACE1 treatment (NeuN neuronal staining coincides withpervasive antibody staining) but absent in control IgG treated mice(only the NeuN neuronal staining pattern is observed, no antibodystaining).

FIGS. 4A-E show that a single systemic dose of anti-TfR^(A)/BACE1significantly reduces central and peripheral Aβ₁₋₄₀. FIGS. 4A-D showquantification of brain (A,B) and plasma (C,D) Aβ₁₋₄₀ levels after a 25mg/kg or 50 mg/kg IV-injection of control IgG, anti-BACE1, oranti-TfR/BACE1. Briefly, for Abeta1-40 measurements, hemi-brains werehomogenized in 5M guanidine hydrochloride buffer and samples rotated for3 hours at room temperature prior to dilution (1:10) in 0.25% casein, 5mM EDTA (pH 8.0) in PBS containing freshly added aprotinin (20 mg/mL)and leupeptin (10 mg/ml). Diluted homogenates were centrifuged at 14,000rpm for 20 min. and supernates were isolated for Abeta1-40 measurement.For antibody concentration measurements, the corresponding hemi-brainfrom each mouse was homogenized in 1% NP-40 as described above. Wholeblood was collected in EDTA microtainer tubes (BD Diagnostics) prior toperfusion, centrifuged at 5,000×g for 15 minutes and the supernatant wasisolated for measuring plasma mouse Abeta 1-40 and anti-TfR/BACE1concentrations. The concentrations of total mouse Abeta1-40 in plasmaand brain were determined using a sandwich ELISA following similarprocedures described above. Rabbit polyclonal antibody specific for theC-terminus of Abeta 1-40 (Millipore, Bedford Mass.) was coated ontoplates, and biotinylated anti-mouse Abeta monoclonal antibody M3.2(Covance, Dedham Mass.) was used for detection. The assay had lowerlimit of quantification values of 1.96 pg/ml in plasma and 39.1 pg/g inbrain. Statistical analysis of differences between experimental groupswas performed using a two-tailed unpaired t-test. * representsignificance compared to control IgG, while # represent significancecompared to anti-BACE1. * p<0.05, ** p<0.01, *** p<0.001; n=10 for allgroups. FIG. 4E shows mean Aβ₁₋₄₀ reduction from data in (A-D)calculated as a percentage of Aβ₁₋₄₀ levels relative to controlIgG-injected mice.

FIGS. 5A-B depict the light and heavy chain amino acid sequences ofanti-BACE1 clone YW412.8 obtained from a naïve sort of the naturaldiversity phage display library and affinity-matured forms of YW412.8.FIG. 5A depicts the variable light (VL) sequence alignments (SEQ ID NOs.1-6). FIG. 5B depicts the variable heavy (VH) sequence alignments (SEQID Nos. 7-8). In both figures, the HVR sequences for each clone areindicated by the boxed regions, with the first box indicating HVR-L1(FIG. 5A) or HVR-H1 (FIG. 5B), the second box indicating HVR-L2 (FIG.5A) or HVR-H2 (FIG. 5B), and the third box indicating HVR-L3 (FIG. 5A)or HVR-H3 (FIG. 5B).

FIGS. 6A-B depict the light and heavy chain amino acid sequences ofclone Fab 12 obtained from a naïve sort of a synthetic diversity phagedisplay library and affinity-matured forms of Fab 12. FIG. 6A depictsthe light chain sequence alignments (SEQ ID NOs. 9-12). FIG. 6B depictsthe heavy chain sequence alignments (SEQ ID NO. 13). In both figures,the HVR sequences for each clone are indicated by the boxed regions,with the first box indicating HVR-L1 (FIG. 6A) or HVR-H1 (FIG. 6B), thesecond box indicating HVR-L2 (FIG. 6A) or HVR-H2 (FIG. 6B), and thethird box indicating HVR-L3 (FIG. 6A) or HVR-H3 (FIG. 6B).

FIGS. 7A-B depict the heavy chain (FIG. 7A; SEQ ID NO. 14) and lightchain (FIG. 7B; SEQ ID NO. 15) of an exemplary anti-Abeta antibody.

FIGS. 8A-B depict the quantification of anti-TfR^(A,B,C,D,E) in theserum (FIG. 8A) and brain (FIG. 8B) after a single therapeutic doseadministration in mice. Six-eight week old wild type female C57B/6 micewere used for all studies. Mice were intravenously injected with 20mg/kg of anti-TfR variants or control IgG. Antibody levels in brain andserum were measured at 1 and 12 hours and 1, 2, 4, 5, 6, and 8 days postinjection. Total injection volume did not exceed 260 μL and antibodieswere diluted in D-PBS (Invitrogen) when necessary. The experiment wasperformed using the same protocol as the experiment whose results areshown in FIG. 1B.

FIGS. 9A-E show the varying degrees to which bispecificanti-TfR^(A,D,E)/BACE1 antibodies accumulate in the brain and inhibit Aβproduction in vivo. FIG. 9A depicts the results of an anti-TfRcompetition ELISA assay using anti-TfR^(A,D,E)/BACE1, following the sameassay procedure as that described in FIG. 2A. The IC50 values determinedfrom this data are shown in Table 3. FIGS. 9B and 9D quantitate theamount of observed antibody (9B) and the amount of Abeta1-40 (9D)observed in the plasma at 1, 2, 4, 6, 8 and 10 days after a 50 mg/kg IVinjection of antibodies in mice (n=6). FIG. 9C depicts quantification ofmean brain uptake and FIG. 9E depicts the amount of Abeta 1-40 observedin the brains of those same treated mice at 1, 2, 4, 6, 8 and 10 daysafter treatment. Six to eight week-old wild type female C57B/6 mice wereused for all studies. Mice were intravenously injected with 50 mg/kganti-TfR/BACE1 variants, control IgG, or anti-BACE1. After the indicatedtime, mice were perfused with D-PBS, and brain and plasma antibodyconcentration for each animal was measured as described above. The assaywas performed as described in the FIG. 4 description.

FIGS. 10 and 11 show the varying degrees to which bispecificanti-TfR^(A,D,E)/Abeta antibodies accumulate in the brain of PS2APP mice(FIG. 12) and wild type mice (FIG. 13). FIGS. 10A and 11A depictquantification of the amount of observed antibody in the plasma 1 dayafter a 50 mg/kg i.p. injection of antibodies in mice (n=4-6). FIGS. 10Band 11B quantitate mean brain uptake in the same treated mice.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. Definitions

The “blood-brain barrier” or “BBB” refers to the physiological barrierbetween the peripheral circulation and the brain and spinal cord whichis formed by tight junctions within the brain capillary endothelialplasma membranes, creating a tight barrier that restricts the transportof molecules into the brain, even very small molecules such as urea (60Daltons). The blood-brain barrier within the brain, the blood-spinalcord barrier within the spinal cord, and the blood-retinal barrierwithin the retina are contiguous capillary barriers within the CNS, andare herein collectively referred to a the blood-brain barrier or BBB.The BBB also encompasses the blood-CSF barrier (choroid plexus) wherethe barrier is comprised of ependymal cells rather than capillaryendothelial cells.

The “central nervous system” or “CNS” refers to the complex of nervetissues that control bodily function, and includes the brain and spinalcord.

A “blood-brain barrier receptor” (abbreviated “BBB-R” herein) is atransmembrane receptor protein expressed on brain endothelial cellswhich is capable of transporting molecules across the blood-brainbarrier. Examples of BBB-R herein include: transferrin receptor (TfR),insulin receptor, insulin-like growth factor receptor (IGF-R), lowdensity lipoprotein receptors including without limitation low densitylipoprotein receptor-related protein 1 (LRP1) and low densitylipoprotein receptor-related protein 8 (LRP8), and heparin-bindingepidermal growth factor-like growth factor (HB-EGF). An exemplary BBB-Rherein is transferrin receptor (TfR).

The “transferrin receptor” (“TfR”) is a transmembrane glycoprotein (witha molecular weight of about 180,000) composed of two disulphide-bondedsub-units (each of apparent molecular weight of about 90,000) involvedin iron uptake in vertebrates. In one embodiment, the TfR herein ishuman TfR comprising the amino acid sequence as in Schneider et al.Nature 311: 675-678 (1984), for example.

A “neurological disorder” as used herein refers to a disease or disorderwhich affects the CNS and/or which has an etiology in the CNS. ExemplaryCNS diseases or disorders include, but are not limited to, neuropathy,amyloidosis, cancer, an ocular disease or disorder, viral or microbialinfection, inflammation, ischemia, neurodegenerative disease, seizure,behavioral disorders, and a lysosomal storage disease. For the purposesof this application, the CNS will be understood to include the eye,which is normally sequestered from the rest of the body by theblood-retina barrier. Specific examples of neurological disordersinclude, but are not limited to, neurodegenerative diseases (including,but not limited to, Lewy body disease, postpoliomyelitis syndrome,Shy-Draeger syndrome, olivopontocerebellar atrophy, Parkinson's disease,multiple system atrophy, striatonigral degeneration, tauopathies(including, but not limited to, Alzheimer disease and supranuclearpalsy), prion diseases (including, but not limited to, bovine spongiformencephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru,Gerstmann-Straussler-Scheinker disease, chronic wasting disease, andfatal familial insomnia), bulbar palsy, motor neuron disease, andnervous system heterodegenerative disorders (including, but not limitedto, Canavan disease, Huntington's disease, neuronalceroid-lipofuscinosis, Alexander's disease, Tourette's syndrome, Menkeskinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome,lafora disease, Rett syndrome, hepatolenticular degeneration,Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia(including, but not limited to, Pick's disease, and spinocerebellarataxia), cancer (e.g. of the CNS and/or brain, including brainmetastases resulting from cancer elsewhere in the body).

A “neurological disorder drug” is a drug or therapeutic agent thattreats one or more neurological disorder(s). Neurological disorder drugsof the invention include, but are not limited to, antibodies, peptides,proteins, natural ligands of one or more CNS target(s), modifiedversions of natural ligands of one or more CNS target(s), aptamers,inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNA) and shorthairpin RNAs (shRNA)), ribozymes, and small molecules, or activefragments of any of the foregoing. Exemplary neurological disorder drugsof the invention are described herein and include, but are not limitedto: antibodies, aptamers, proteins, peptides, inhibitory nucleic acidsand small molecules and active fragments of any of the foregoing thateither are themselves or specifically recognize and/or act upon (i.e.,inhibit, activate, or detect) a CNS antigen or target molecule such as,but not limited to, amyloid precursor protein or portions thereof,amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein,parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancermarkers, and neurotrophins. Non-limiting examples of neurologicaldisorder drugs and disorders they may be used to treat are provided inthe following Table 1:

TABLE 1 Non-limiting examples of neurological disorder drugs and thecorresponding disorders they may be used to treat Drug Neurologicaldisorder Anti-BACE1 Antibody Alzheimer's, acute and chronic braininjury, stroke Anti-Abeta Antibody Alzheimer's disease NeurotrophinStroke, acute brain injury, spinal cord injury Brain-derivedneurotrophic factor Chronic brain injury (BDNF), Fibroblast growth(Neurogenesis) factor 2 (FGF-2) Anti-Epidermal Growth Factor ReceptorBrain cancer (EGFR)-antibody Glial cell-line derived neural factorParkinson's disease (GDNF) Brain-derived neurotrophic factor (BDNF)Amyotrophic lateral sclerosis, depression Lysosomal enzyme Lysosomalstorage disorders of the brain Ciliary neurotrophic factor (CNTF)Amyotrophic lateral sclerosis Neuregulin-1 Schizophrenia Anti-HER2antibody (e.g. trastuzumab) Brain metastasis from HER2-positive cancer

An “imaging agent” is a compound that has one or more properties thatpermit its presence and/or location to be detected directly orindirectly. Examples of such imaging agents include proteins and smallmolecule compounds incorporating a labeled moiety that permitsdetection.

A “CNS antigen” or “brain antigen” is an antigen expressed in the CNS,including the brain, which can be targeted with an antibody or smallmolecule. Examples of such antigens include, without limitation:beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factorreceptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau,apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6. In one embodiment, the antigen is BACE1.

The term “BACE1,” as used herein, refers to any native beta-secretase 1(also called β-site amyloid precursor protein cleaving enzyme 1,membrane-associated aspartic protease 2, memapsin 2, aspartyl protease 2or Asp2) from any vertebrate source, including mammals such as primates(e.g. humans) and rodents (e.g., mice and rats), unless otherwiseindicated. The term encompasses “full-length,” unprocessed BACE1 as wellas any form of BACE1 which results from processing in the cell. The termalso encompasses naturally occurring variants of BACE1, e.g., splicevariants or allelic variants. The amino acid sequence of an exemplaryBACE1 polypeptide is the sequence for human BACE1, isoform A as reportedin Vassar et al., Science 286:735-741 (1999), which is incorporatedherein by reference in its entirety. Several other isoforms of humanBACE1 exist including isoforms B, C and D. See UniProtKB/Swiss-ProtEntry P56817, which is incorporated herein by reference in its entirety.

The terms “anti-beta-secretase antibody”, “anti-BACE1 antibody”, “anantibody that binds to beta-secretase” and “an antibody that binds toBACE1” refer to an antibody that is capable of binding BACE1 withsufficient affinity such that the antibody is useful as a diagnosticand/or therapeutic agent in targeting BACE1. In one embodiment, theextent of binding of an anti-BACE1 antibody to an unrelated, non-BACE1protein is less than about 10% of the binding of the antibody to BACE1as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments,an antibody that binds to BACE1 has a dissociation constant (Kd) of ≦1μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸Mor less, e.g. from 10⁻⁸ M to H⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). Incertain embodiments, an anti-BACE1 antibody binds to an epitope of BACE1that is conserved among BACE1 from different species and isoforms. Inone embodiment, an antibody is provided that binds to the epitope onBACE1 bound by anti-BACE1 antibody YW412.8.31. In other embodiments, anantibody is provided that binds to an exosite within BACE1 located inthe catalytic domain of BACE1. In one embodiment an antibody is providedthat competes with the peptides identified in Kornacker et al., Biochem.44:11567-11573 (2005), which is incorporated herein by reference in itsentirety, (i.e., Peptides 1, 2, 3, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 2-12,3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, 4, 5, 6, 5-10, 5-9,scrambled, Y5A, P6A, Y7A, FSA, 19A, P10A and L11A) for binding to BACE1.Exemplary BACE1 antibody sequences are depicted in FIG. 5A-B and FIG.6A-B. One exemplary antibody herein comprises the variable domains ofthe antibody YW412.8.31 (e.g. as in FIGS. 5A-B).

A “native sequence” protein herein refers to a protein comprising theamino acid sequence of a protein found in nature, including naturallyoccurring variants of the protein. The term as used herein includes theprotein as isolated from a natural source thereof or as recombinantlyproduced.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments so long as theyexhibit the desired biological activity.

“Antibody fragments” herein comprise a portion of an intact antibodywhich retains the ability to bind antigen. Examples of antibodyfragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies;linear antibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variants that mayarise during production of the monoclonal antibody, such variantsgenerally being present in minor amounts. In contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Inaddition to their specificity, the monoclonal antibodies areadvantageous in that they are uncontaminated by other immunoglobulins.The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.Biol., 222:581-597 (1991), for example. Specific examples of monoclonalantibodies herein include chimeric antibodies, humanized antibodies, andhuman antibodies, including antigen-binding fragments thereof.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).Chimeric antibodies of interest herein include “primatized” antibodiescomprising variable domain antigen-binding sequences derived from anon-human primate (e.g. Old World Monkey, such as baboon, rhesus orcynomolgus monkey) and human constant region sequences (U.S. Pat. No.5,693,780).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence, except for FR substitution(s) as noted above. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region, typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992).

A “human antibody” herein is one comprising an amino acid sequencestructure that corresponds with the amino acid sequence structure of anantibody obtainable from a human B-cell, and includes antigen-bindingfragments of human antibodies. Such antibodies can be identified or madeby a variety of techniques, including, but not limited to: production bytransgenic animals (e.g., mice) that are capable, upon immunization, ofproducing human antibodies in the absence of endogenous immunoglobulinproduction (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos.5,591,669, 5,589,369 and 5,545,807)); selection from phage displaylibraries expressing human antibodies or human antibody fragments (see,for example, McCafferty et al., Nature 348:552-553 (1990); Johnson etal., Current Opinion in Structural Biology 3:564-571 (1993); Clackson etal., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597(1991); Griffith et al., EMBO J. 12:725-734 (1993); U.S. Pat. Nos.5,565,332 and 5,573,905); generation via in vitro activated B cells (seeU.S. Pat. Nos. 5,567,610 and 5,229,275); and isolation from humanantibody producing hybridomas.

A “multispecific antibody” herein is an antibody having bindingspecificities for at least two different epitopes. Exemplarymultispecific antibodies may bind both a BBB-R and a brain antigen.Multispecific antibodies can be prepared as full-length antibodies orantibody fragments (e.g. F(ab′)₂ bispecific antibodies). Engineeredantibodies with two, three or more (e.g. four) functional antigenbinding sites are also, contemplated (see, e.g., US Appln No. US2002/0004587 A1, Miller et al.). Multispecific antibodies can beprepared as full length antibodies or antibody fragments.

Antibodies herein include “amino acid sequence variants” with alteredantigen-binding or biological activity. Examples of such amino acidalterations include antibodies with enhanced affinity for antigen (e.g.“affinity matured” antibodies), and antibodies with altered Fc region,if present, e.g. with altered (increased or diminished) antibodydependent cellular cytotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) (see, for example, WO 00/42072, Presta, L. and WO99/51642, Iduosogie et al.); and/or increased or diminished serumhalf-life (see, for example, WO00/42072, Presta, L.).

An “affinity modified variant” has one or more substituted hypervariableregion or framework residues of a parent antibody (e.g. of a parentchimeric, humanized, or human antibody) that alter (increase or reduce)affinity. In one embodiment, the resulting variant(s) selected forfurther development will have reduced affinity for the BBB-R accordingto the present invention. A convenient way for generating suchsubstitutional variants uses phage display. Briefly, severalhypervariable region sites (e.g. 6-7 sites) are mutated to generate allpossible amino substitutions at each site. The antibody variants thusgenerated are displayed in a monovalent fashion from filamentous phageparticles as fusions to the gene III product of M13 packaged within eachparticle. The phage-displayed variants are then screened for theirbiological activity (e.g. binding affinity). In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand its target. Such contact residues and neighboring residues arecandidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening and antibodies with altered affinity may beselected for further development.

The antibody herein may be conjugated with a “heterologous molecule” forexample to increase half-life or stability or otherwise improve theantibody. For example, the antibody may be linked to one of a variety ofnon-proteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, polyoxyalkylenes, or copolymers of polyethyleneglycol and polypropylene glycol. Antibody fragments, such as Fab′,linked to one or more PEG molecules are an exemplary embodiment of theinvention.

The antibody herein may be a “glycosylation variant” such that anycarbohydrate attached to the Fc region, if present, is altered. Forexample, antibodies with a mature carbohydrate structure that lacksfucose attached to an Fc region of the antibody are described in US PatAppl No US 2003/0157108 (Presta, L.). See also US 2004/0093621 (KyowaHakko Kogyo Co., Ltd). Antibodies with a bisecting N-acetylglucosamine(GlcNAc) in the carbohydrate attached to an Fc region of the antibodyare referenced in WO 2003/011878, Jean-Mairet et al. and U.S. Pat. No.6,602,684, Umana et al. Antibodies with at least one galactose residuein the oligosaccharide attached to an Fc region of the antibody arereported in WO 1997/30087, Patel et al. See, also, WO 1998/58964 (Raju,S.) and WO 1999/22764 (Raju, S.) concerning antibodies with alteredcarbohydrate attached to the Fc region thereof. See also US 2005/0123546(Umana et al.) describing antibodies with modified glycosylation.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain;Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “Framework”or “FR” residues are those variable domain residues other than thehypervariable region residues as herein defined.

A “full length antibody” is one which comprises an antigen-bindingvariable region as well as a light chain constant domain (CL) and heavychain constant domains, CH1, CH2 and CH3. The constant domains may benative sequence constant domains (e.g. human native sequence constantdomains) or amino acid sequence variants thereof.

A “naked antibody” is an antibody (as herein defined) that is notconjugated to a heterologous molecule, such as a cytotoxic moiety,polymer, or radiolabel.

Antibody “effector functions” refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include Clq binding, complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), etc. In one embodiment, the antibodyherein essentially lacks effector function.

Depending on the amino acid sequence of the constant domain of theirheavy chains, full length antibodies can be assigned to different“classes”. There are five major classes of full length antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The term “recombinant antibody”, as used herein, refers to an antibody(e.g. a chimeric, humanized, or human antibody or antigen-bindingfragment thereof) that is expressed by a recombinant host cellcomprising nucleic acid encoding the antibody. Examples of “host cells”for producing recombinant antibodies include: (1) mammalian cells, forexample, Chinese Hamster Ovary (CHO), COS, myeloma cells (including Y0and NS0 cells), baby hamster kidney (BHK), Hela and Vero cells; (2)insect cells, for example, sf9, sf21 and Tn5; (3) plant cells, forexample plants belonging to the genus Nicotiana (e.g. Nicotianatabacum); (4) yeast cells, for example, those belonging to the genusSaccharomyces (e.g. Saccharomyces cerevisiae) or the genus Aspergillus(e.g. Aspergillus niger); (5) bacterial cells, for example Escherichia.coli cells or Bacillus subtilis cells, etc.

As used herein, “specifically binding” or “binds specifically to” refersto an antibody selectively or preferentially binding to an antigen. Thebinding affinity is generally determined using a standard assay, such asScatchard analysis, or surface plasmon resonance technique (e.g. usingBIACORE®).

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. In one embodiment, an anti-BACE1antibody binds to the BACE1 epitope bound by YW412.8.31.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An “immunoconjugate” is an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a label orcytotoxic agent. Optionally such conjugation is via a linker.

A “linker” as used herein is a structure that covalently ornon-covalently connects the anti-BBB-R antibody to heterologousmolecule. In certain embodiments, a linker is a peptide. In otherembodiments, a linker is a chemical linker.

A “label” is a marker coupled with the antibody herein and used fordetection or imaging. Examples of such labels include: radiolabel, afluorophore, a chromophore, or an affinity tag. In one embodiment, thelabel is a radiolabel used for medical imaging, for example tc99m orI123, or a spin label for nuclear magnetic resonance (NMR) imaging (alsoknown as magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese, iron, etc.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chroinatogr. B 848:79-87 (2007).

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

II. Compositions and Methods

II. Production of Anti-BBB-R Antibodies and Conjugates Thereof

The methods and articles of manufacture of the present invention use, orincorporate, an antibody that binds to BBB-R. The BBB-R antigen to beused for production of, or screening for, antibodies may be, e.g., asoluble form of or a portion thereof (e.g. the extracellular domain),containing the desired epitope. Alternatively, or additionally, cellsexpressing BBB-R at their cell surface can be used to generate, orscreen for, antibodies. Other forms of BBB-R useful for generatingantibodies will be apparent to those skilled in the art. Examples ofBBB-Rs herein include transferrin receptor (TfR), insulin receptor,insulin-like growth factor receptor (IGF-R), low density lipoproteinreceptor-related protein 1 (LRP1) and LRP8 etc, and heparin-bindingepidermal growth factor-like growth factor (HB-EGF). According to thepresent invention, a “low affinity” anti-BBB-R (e.g. anti-TfR) antibodyis selected based on the data herein demonstrating that such antibodiesdisplay improved CNS (for example, brain) uptake. In order to identifysuch low affinity antibodies, various assays for measuring antibodyaffinity are available including, without limitation: Scatchard assayand surface plasmon resonance technique (e.g. using BIACORE®). Accordingto one embodiment of the invention, the antibody has an affinity for theBBB-R antigen (e.g. for TfR) from about 5 nM, or from about 20 nM, orfrom about 100 nM, to about 10 μM, or to about 1 μM, or to about 500 nM.Thus, the affinity may be in the range from about 5 nM to about 10 μM,or in the range from about 20 nM to about 1 μM, or in the range fromabout 100 nM to about 500 nM, e.g. as measured by Scatchard analysis orBIACORE®.

Thus, the invention provides a method of making an antibody useful fortransporting a neurological disorder drug across the blood-brain barriercomprising selecting an antibody from a panel of antibodies against ablood-brain barrier receptor (BBB-R) because it has an affinity for theBBB-R which is in the range from about 5 nM, or from about 20 nM, orfrom about 100 nM, to about 10 μM, or to about 1 μM, or to about 500 mM.Thus, the affinity may be in the range from about 5 nM to about 10 μM orin the range from about 20 nM to about 1 μM, or in the range from about100 nM to about 500 nM, e.g. as measured by Scatchard analysis orBIACORE®. As will be understood by one of ordinary skill in the art,conjugating a heterologous molecule/compound to an antibody will oftendecrease the affinity of the antibody for its target due, e.g., tosteric hindrance or even to elimination of one binding arm if theantibody is made multispecific with one or more arms binding to adifferent antigen than the antibody's original target. In oneembodiment, a low affinity antibody of the invention specific for TfRconjugated to BACE1 had a Kd for TfR as measured by BIACORE of about 30nM. In another embodiment, a low affinity antibody of the inventionspecific for TfR conjugated to BACE1 had a Kd for TfR as measured byBIACORE of about 600 nM.

One exemplary assay for evaluating antibody affinity is by Scatchardanalysis. For example, the anti-BBB-R antibody of interest can beiodinated using the lactoperoxidase method (Bennett and Horuk, Methodsin Enzymology 288 pg. 134-148 (1997)). A radiolabeled anti-BBB-Rantibody is purified from free ¹²⁵I-Na by gel filtration using a NAP-5column and its specific activity measured. Competition reaction mixturesof 50 μL containing a fixed concentration of iodinated antibody anddecreasing concentrations of serially diluted unlabeled antibody areplaced into 96-well plates. Cells transiently expressing BBB-R arecultured in growth media, consisting of Dulbecco's modified eagle'smedium (DMEM) (Genentech) supplemented with 10% FBS, 2 mM L-glutamineand 1× penicillin-streptomycin at 37° C. in 5% CO₂. Cells are detachedfrom the dishes using Sigma Cell Dissociation Solution and washed withbinding buffer (DMEM with 1% bovine serum albumin, 50 mM HEPES, pH 7.2,and 0.2% sodium azide). The washed cells are added at an approximatedensity of 200,000 cells in 0.2 mL of binding buffer to the 96-wellplates containing the 50-μL competition reaction mixtures. The finalconcentration of the unlabeled antibody in the competition reaction withcells is varied, starting at 1000 nM and then decreasing by 1:2 folddilution for 10 concentrations and including a zero-added, buffer-onlysample. Competition reactions with cells for each concentration ofunlabeled antibody are assayed in triplicate. Competition reactions withcells are incubated for 2 hours at room temperature. After the 2-hourincubation, the competition reactions are transferred to a filter plateand washed four times with binding buffer to separate free from boundiodinated antibody. The filters are counted by gamma counter and thebinding data are evaluated using the fitting algorithm of Munson andRodbard (1980) to determine the binding affinity of the antibody.

An exemplary scatchard analysis using the compositions of the inventionmay be performed as follows. Anti-TFR^(A) was iodinated using thelactoperoxidase method (Bennett and Horuk, Methods in Enzymology 288 pg.134-148 (1997)). Radiolabeled anti-TFR^(A) was purified from free¹²⁵I-Na by gel filtration using a NAP-5 column; purified anti-TFR^(A)had a specific activity of 19.82 μCi/μg. Competition reaction mixturesof 50 μL containing a fixed concentration of iodinated antibody anddecreasing concentrations of serially diluted unlabeled antibody wereplaced into 96-well plates. The 293 cells transiently expressing murineTfR were cultured in growth media, consisting of Dulbecco's modifiedeagle's medium (DMEM) (Genentech) supplemented with 10% FBS, 2 mML-glutamine and 1× penicillin-streptomycin at 37° C. in 5% CO₂. Cellswere detached from the dishes using Sigma Cell Dissociation Solution andwashed with binding buffer (DMEM with 1% bovine serum albumin, 50 mMHEPES, pH 7.2, and 0.2% sodium azide). The washed cells were added at anapproximate density of 200,000 cells in 0.2 mL of binding buffer to the96-well plates containing the 50-μL competition reaction mixtures. Thefinal concentration of the iodinated antibody in each competitionreaction with cells was 100 μM (134,000 cpm per 0.25 mL). The finalconcentration of the unlabeled antibody in the competition reaction withcells varied, starting at 1000 nM and then decreasing by 1:2 folddilution for 10 concentrations and including a zero-added, buffer-onlysample. Competition reactions with cells for each concentration ofunlabeled antibody were assayed in triplicate. Competition reactionswith cells were incubated for 2 hours at room temperature. After the2-hour incubation, the competition reactions were transferred to aMillipore Multiscreen filter plate and washed four times with bindingbuffer to separate free from bound iodinated antibody. The filters werecounted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life andAnalytical Sciences; Waltham, Mass.). The binding data were evaluatedusing New Ligand software (Genentech), which uses the fitting algorithmof Munson and Rodbard (1980) to determine the binding affinity of theantibody.

An exemplary BIACORE® analysis using the compositions of the inventionmay be performed as follows. Kd was measured using surface plasmonresonance assays using a BIACORE®-2000 (BIAcore, Inc., Piscataway, N.J.)at 25° C. using anti-human Fc kit (BiAcore Inc., Piscataway, N.J.).Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE, Inc.)were activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-human Fc antibody was diluted with 10 mMsodium acetate, pH 4.0, to 50 pg/ml before injection at a flow rate of 5μl/minute to achieve approximately 10000 response units (RU) of coupledprotein. Following the injection of antibody, 1 M ethanolamine wasinjected to block unreacted groups. For kinetics measurements, anti-TfRantibody variants were injected in HBS-P to reach about 220 RU, thentwo-fold serial dilutions of MuTfR-His (0.61 nM to 157 nM) were injectedin HBS-P at 25° C. at a flow rate of approximately 30 μl/min.Association rates (kon) and dissociation rates (koff) were calculatedusing a simple one-to-one Langmuir binding model (BIACORE® EvaluationSoftware version 3.2) by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (Kd) wascalculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999)

According to another embodiment, Kd is measured using surface plasmonresonance assays with a BIACORE®-2000 device (BIAcore, Inc., Piscataway,N.J.) at 25° C. using anti-human Fc kit (BiAcore Inc., Piscataway,N.J.). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE,Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Anti-human Fc antibody is diluted with 10 mMsodium acetate, pH 4.0, to 50 μg/ml before injection at a flow rate of 5μl/minute to achieve approximately 10000 response units (RU) of coupledprotein. Following the injection of antibody, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements,anti-BBB-R antibody variants are injected in HBS-P to reach about 220RU, then two-fold serial dilutions of BBB-R-His (0.61 nM to 157 nM) areinjected in HBS-P at 25° C. at a flow rate of approximately 30 μl/min.Association rates (kon) and dissociation rates (koff) are calculatedusing a simple one-to-one Langmuir binding model (BIACORE® EvaluationSoftware version 3.2) by simultaneously fitting the association anddissociation sensorgrams. The equilibrium dissociation constant (Kd) iscalculated as the ratio koff/kon. See, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999).

A surrogate measurement for the affinity of one or more antibodies forthe BBB-R is its half maximal inhibitory concentration (IC50), a measureof how much of the antibody is needed to inhibit the binding of a knownBBB-R ligand to the BBB-R by 50%. Several methods of determining theIC50 for a given compound are art-known; a common approach is to performa competition binding assay, such as that described herein in theexamples, i.e. with regard to FIG. 2A. In general, a high IC50 indicatesthat more of the antibody is required to inhibit binding of the knownligand, and thus that the antibody's affinity for that ligand isrelatively low. Conversely, a low IC50 indicates that less of theantibody is required to inhibit binding of the known ligand, and thusthat the antibody's affinity for that ligand is relatively high.

An exemplary competitive ELISA assay to measure IC50 is one in whichincreasing concentrations of anti-TfR or anti-TfR/brain antigen (i.e.,anti-TfR/BACE1 anti-TfR/Abeta and the like) variant antibodies are usedto compete against biotinylated TfR^(A) for binding to TfR. The anti-TfRcompetition ELISA was performed in Maxisorp plates (Neptune, N.J.)coated with 2.5 μg/ml of purified murine TfR extracellular domain in PBSat 4° C. overnight. Plates were washed with PBS/0.05% Tween 20 andblocked using Superblock blocking buffer in PBS (Thermo Scientific,Hudson, N.H.). A titration of each individual anti-TfR or anti-TfR/brainantigen (i.e., anti-TfR/BACE1 or anti-TfR/Abeta) (1:3 serial dilution)was combined with biotinylated anti-TfR^(A) (0.5 nM final concentration)and added to the plate for 1 hour at room temperature. Plates werewashed with PBS/0.05% Tween 20, and HRP-streptavidin (Southern Biotech,Birmingham) was added to the plate and incubated for 1 hour at roomtemperature. Plates were washed with PBS/0.05% Tween 20, andbiotinylated anti-TfR^(A) bound to the plate was detected using TMBsubstrate (BioFX Laboratories, Owings Mills).

In one embodiment, the low affinity anti-BBB-R antibody herein iscoupled with a label and/or neurological disorder drug or imaging agentin order to more efficiently transport the label and/or drug or imagingagent across the BBB. Such coupling can be achieved by chemicalcross-linkers or by generating fusion proteins etc.

Covalent conjugation can either be direct or via a linker. In certainembodiments, direct conjugation is by construction of a protein fusion(i.e., by genetic fusion of the two genes encoding BBB-R antibody andneurological disorder drug and expression as a single protein). Incertain embodiments, direct conjugation is by formation of a covalentbond between a reactive group on one of the two portions of theanti-BBB-R antibody and a corresponding group or acceptor on theneurological drug. In certain embodiments, direct conjugation is bymodification (i.e., genetic modification) of one of the two molecules tobe conjugated to include a reactive group (as nonlimiting examples, asulfhydryl group or a carboxyl group) that forms a covalent attachmentto the other molecule to be conjugated under appropriate conditions. Asone nonlimiting example, a molecule (i.e., an amino acid) with a desiredreactive group (i.e., a cysteine residue) may be introduced into, e.g.,the anti-BBB-R antibody and a disulfide bond formed with theneurological drug. Methods for covalent conjugation of nucleic acids toproteins are also known in the art (i.e., photocrosslinking, see, e.g.,Zatsepin et al. Russ. Chem. Rev. 74: 77-95 (2005)) Non-covalentconjugation can be by any nonconvalent attachment means, includinghydrophobic bonds, ionic bonds, electrostatic interactions, and thelike, as will be readily understood by one of ordinary skill in the art.Conjugation may also be performed using a variety of linkers. Forexample, an anti-BBB-R antibody and a neurological drug may beconjugated using a variety of bifunctional protein coupling agents suchas N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Peptide linkers, comprised of from oneto twenty amino acids joined by peptide bonds, may also be used. Incertain such embodiments, the amino acids are selected from the twentynaturally-occurring amino acids. In certain other such embodiments, oneor more of the amino acids are selected from glycine, alanine, proline,asparagine, glutamine and lysine. The linker may be a “cleavable linker”facilitating release of the neurological drug upon delivery to thebrain. For example, an acid-labile linker, peptidase-sensitive linker,photolabile linker, dimethyl linker or disulfide-containing linker(Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020)may be used.

The invention herein expressly contemplates, but is not limited to,conjugates prepared with cross-linker reagents including, but notlimited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB,SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS,sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

For a neuropathy disorder, a neurological drug may be selected that isan analgesic including, but not limited to, a narcotic/opioid analgesic(i.e., morphine, fentanyl, hydrocodone, meperidine, methadone,oxymorphone, pentazocine, propoxyphene, tramadol, codeine andoxycodone), a nonsteroidal anti-inflammatory drug (NSAID) (i.e.,ibuprofen, naproxen, diclofenac, diflunisal, etodolac, fenoprofen,flurbiprofen, indomethacin, ketorolac, mefenamic acid, meloxicam,nabumetone, oxaprozin, piroxicam, sulindac, and tolmetin), acorticosteroid (i.e., cortisone, prednisone, prednisolone,dexamethasone, methylprednisolone and triamcinolone), an anti-migraineagent (i.e., sumatriptin, almotriptan, frovatriptan, sumatriptan,rizatriptan, eletriptan, zolmitriptan, dihydroergotamine, eletriptan andergotamine), acetaminophen, a salicylate (i.e., aspirin, cholinesalicylate, magnesium salicylate, diflunisal, and salsalate), aanti-convulsant (i.e., carbamazepine, clonazepam, gabapentin,lamotrigine, pregabalin, tiagabine, and topiramate), an anaesthetic(i.e., isoflurane, trichloroethylene, halothane, sevoflurane,benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine,propoxycaine, procaine, novocaine, proparacaine, tetracaine, articaine,bupivacaine, carticaine, cinchocaine, etidocaine, levobupivacaine,lidocaine, mepivacaine, piperocaine, prilocalne, ropivacaine,trimecaine, saxitoxin and tetrodotoxin), and a cox-2-inhibitor (i.e.,celecoxib, rofecoxib, and valdecoxib). For a neuropathy disorder withvertigo involvement, a neurological drug may be selected that is ananti-vertigo agent including, but not limited to, meclizine,diphenhydramine, promethazine and diazepam. For a neuropathy disorderwith nausea involvement, a neurological drug may be selected that is ananti-nausea agent including, but not limited to, promethazine,chlorpromazine, prochlorperazine, trimethobenzamide, and metoclopramide.For a neurodegenerative disease, a neurological drug may be selectedthat is a growth hormone or neurotrophic factor; examples include butare not limited to brain-derived neurotrophic factor (BDNF), nervegrowth factor (NGF), neurotrophin-4/5, fibroblast growth factor (FGF)-2and other FGFs, neurotrophin (NT)-3, erythropoietin (EPO), hepatocytegrowth factor (HGF), epidermal growth factor (EGF), transforming growthfactor (TGF)-alpha, TGF-beta, vascular endothelial growth factor (VEGF),interleukin-1 receptor antagonist (IL-1ra), ciliary neurotrophic factor(CNTF), glial-derived neurotrophic factor (GDNF), neurturin,platelet-derived growth factor (PDGF), heregulin, neuregulin, artemin,persephin, interleukins, glial cell line derived neurotrophic factor(GFR), granulocyte-colony stimulating factor (CSF),granulocyte-macrophage-CSF, netrins, cardiotrophin-1, hedgehogs,leukemia inhibitory factor (LIF), midkine, pleiotrophin, bonemorphogenetic proteins (BMPs), netrins, saposins, semaphorins, and stemcell factor (SCF).

For cancer, a neurological drug may be selected that is achemotherapeutic agent. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphor-amide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antiobiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenishes such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition of chemotherapeutic agents areanti-hormonal agents that act to regulate, reduce, block, or inhibit theeffects of hormones that can promote the growth of cancer, and are oftenin the form of systemic, or whole-body treatment. They may be hormonesthemselves. Examples include anti-estrogens and selective estrogenreceptor modulators (SERMs), including, for example, tamoxifen(including NOLVADEX® tamoxifen), EVISTA® raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; anti-progesterones; estrogen receptordown-regulators (ERDs); agents that function to suppress or shut downthe ovaries, for example, leutinizing hormone-releasing hormone (LHRH)agonists such as LUPRON® and ELIGARD® leuprolide acetate, goserelinacetate, buserelin acetate and tripterelin; other anti-androgens such asflutamide, nilutamide and bicalutamide; and aromatase inhibitors thatinhibit the enzyme aromatase, which regulates estrogen production in theadrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole. In addition, such definition of chemotherapeuticagents includes bisphosphonates such as clodronate (for example,BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronicacid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate, SKELID®tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those that inhibit expression of genes in signalingpathways implicated in aberrant cell proliferation, such as, forexample, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines,for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID®vaccine; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH;lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinasesmall-molecule inhibitor also known as GW572016); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above.

Another group of compounds that may be selected as neurological drugsfor cancer treatment or prevention are anti-cancer immunoglobulins(including, but not limited to, trastuzumab, bevacizumab, alemtuxumab,cetuximab, gemtuzumab ozogamicin, ibritumomab tiuxetan, panitumumab andrituximab). In some instances, antibodies in conjunction with a toxiclabel may be used to target and kill desired cells (i.e., cancer cells),including, but not limited to, tositumomab with a ¹³¹I radiolabel.

For an ocular disease or disorder, a neurological drug may be selectedthat is an anti-angiogenic ophthalmic agent (i.e., bevacizumab,ranibizumab and pegaptanib), an ophthalmic glaucoma agent (i.e.,carbachol, epinephrine, demecarium bromide, apraclonidine, brimonidine,brinzolamide, levobunolol, timolol, betaxolol, dorzolamide, bimatoprost,carteolol, metipranolol, dipivefrin, travoprost and latanoprost), acarbonic anhydrase inhibitor (i.e., methazolamide and acetazolamide), anophthalmic antihistamine (i.e., naphazoline, phenylephrine andtetrahydrozoline), an ocular lubricant, an ophthalmic steroid (i.e.,fluorometholone, prednisolone, loteprednol, dexamethasone,difluprednate, rimexolone, fluocinolone, medrysone and triamcinolone),an ophthalmic anesthetic (i.e., lidocaine, proparacaine and tetracaine),an ophthalmic anti-infective (i.e., levofloxacin, gatifloxacin,ciprofloxacin, moxifloxacin, chloramphenicol, bacitracin/polymyxin b,sulfacetamide, tobramycin, azithromycin, besifloxacin, norfloxacin,sulfisoxazole, gentamicin, idoxuridine, erythromycin, natamycin,gramicidin, neomycin, ofloxacin, trifluridine, ganciclovir, vidarabine),an ophthalmic anti-inflammatory agent (i.e., nepafenac, ketorolac,flurbiprofen, suprofen, cyclosporine, triamcinolone, diclofenac andbromfenac), and an ophthalmic antihistamine or decongestant (i.e.,ketotifen, olopatadine, epinastine, naphazoline, cromolyn,tetrahydrozoline, pemirolast, bepotastine, naphazoline, phenylephrine,nedocromil, lodoxamide, phenylephrine, emedastine and azelastine).

For a seizure disorder, a neurological drug may be selected that is ananticonvulsant or antiepileptic including, but not limited to,barbiturate anticonvulsants (i.e., primidone, metharbital,mephobarbital, allobarbital, amobarbital, aprobarbital, alphenal,barbital, brallobarbital and phenobarbital), benzodiazepineanticonvulsants (i.e., diazepam, clonazepam, and lorazepam), carbamateanticonvulsants (i.e. felbamate), carbonic anhydrase inhibitoranticonvulsants (i.e., acetazolamide, topiramate and zonisamide),dibenzazepine anticonvulsants (i.e., rufinamide, carbamazepine, andoxcarbazepine), fatty acid derivative anticonvulsants (i.e., divalproexand valproic acid), gamma-aminobutyric acid analogs (i.e., pregabalin,gabapentin and vigabatrin), gamma-aminobutyric acid reuptake inhibitors(i.e., tiagabine), gamma-aminobutyric acid transaminase inhibitors(i.e., vigabatrin), hydantoin anticonvulsants (i.e. phenyloin, ethotoin,fosphenyloin and mephenyloin), miscellaneous anticonvulsants (i.e.,lacosamide and magnesium sulfate), progestins (i.e., progesterone),oxazolidinedione anticonvulsants (i.e., paramethadione andtrimethadione), pyrrolidine anticonvulsants (i.e., levetiracetam),succinimide anticonvulsants (i.e., ethosuximide and methsuximide),triazine anticonvulsants (i.e., lamotrigine), and urea anticonvulsants(i.e., phenacemide and pheneturide).

For a lysosomal storage disease, a neurological drug may be selectedthat is itself or otherwise mimics the activity of the enzyme that isimpaired in the disease. Exemplary recombinant enzymes for the treatmentof lysosomal storage disorders include, but are not limited to those setforth in e.g., U.S. Patent Application publication no. 2005/0142141(i.e., alpha-L-iduronidase, iduronate-2-sulphatase, N-sulfatase,alpha-N-acetylglucosaminidase, N-acetyl-galactosamine-6-sulfatase,beta-galactosidase, arylsulphatase B, beta-glucuronidase, acidalpha-glucosidase, glucocerebrosidase, alpha-galactosidase A,hexosaminidase A, acid sphingomyelinase, beta-galactocerebrosidase,beta-galactosidase, arylsulfatase A, acid ceramidase, aspartoacylase,palmitoyl-protein thioesterase 1 and tripeptidyl amino peptidase 1).

For amyloidosis, a neurological drug may be selected that includes, butis not limited to, an antibody or other binding molecule (including, butnot limited to a small molecule, a peptide, an aptamer, or other proteinbinder) that specifically binds to a target selected from: betasecretase, tau, presenilin, amyloid precursor protein or portionsthereof, amyloid beta peptide or oligomers or fibrils thereof, deathreceptor 6 (DR6), receptor for advanced glycation endproducts (RAGE),parkin, and huntingtin; a cholinesterase inhibitor (i.e., galantamine,donepezil, rivastigmine and tacrine); an NMDA receptor antagonist (i.e.,memantine), a monoamine depletor (i.e., tetrabenazine); an ergoloidmesylate; an anticholinergic antiparkinsonism agent (i.e., procyclidine,diphenhydramine, trihexylphenidyl, benztropine, biperiden andtrihexyphenidyl); a dopaminergic antiparkinsonism agent (i.e.,entacapone, selegiline, pramipexole, bromocriptine, rotigotine,selegiline, ropinirole, rasagiline, apomorphine, carbidopa, levodopa,pergolide, tolcapone and amantadine); a tetrabenazine; ananti-inflammatory (including, but not limited to, a nonsteroidalanti-inflammatory drug (i.e., indomethicin and other compounds listedabove); a hormone (i.e., estrogen, progesterone and leuprolide); avitamin (i.e., folate and nicotinamide); a dimebolin; a homotaurine(i.e., 3-aminopropanesulfonic acid; 3APS); a serotonin receptor activitymodulator (i.e., xaliproden); an, an interferon, and a glucocorticoid.For a viral or microbial disease, a neurological drug may be selectedthat includes, but is not limited to, an antiviral compound (including,but not limited to, an adamantane antiviral (i.e., rimantadine andamantadine), an antiviral interferon (i.e., peginterferon alfa-2b), achemokine receptor antagonist (i.e., maraviroc), an integrase strandtransfer inhibitor (i.e., raltegravir), a neuraminidase inhibitor (i.e.,oseltamivir and zanamivir), a non-nucleoside reverse transcriptaseinhibitor (i.e., efavirenz, etravirine, delavirdine and nevirapine), anucleoside reverse transcriptase inhibitors (tenofovir, abacavir,lamivudine, zidovudine, stavudine, entecavir, emtricitabine, adefovir,zalcitabine, telbivudine and didanosine), a protease inhibitor (i.e.,darunavir, atazanavir, fosamprenavir, tipranavir, ritonavir, nelfinavir,amprenavir, indinavir and saquinavir), a purine nucleoside (i.e.,valacyclovir, famciclovir, acyclovir, ribavirin, ganciclovir,valganciclovir and cidofovir), and a miscellaneous antiviral (i.e.,enfuvirtide, foscarnet, palivizumab and fomivirsen)), an antibiotic(including, but not limited to, an aminopenicillin (i.e., amoxicillin,ampicillin, oxacillin, nafcillin, cloxacillin, dicloxacillin,flucoxacillin, temocillin, azlocillin, carbenicillin, ticarcillin,mezlocillin, piperacillin and bacampicillin), a cephalosporin (i.e.,cefazolin, cephalexin, cephalothin, cefamandole, ceftriaxone,cefotaxime, cefpodoxime, ceftazidime, cefadroxil, cephradine,loracarbef, cefotetan, cefuroxime, cefprozil, cefaclor, and cefoxitin),a carbapenem/penem (i.e., imipenem, meropenem, ertapenem, faropenem anddoripenem), a monobactam (i.e., aztreonam, tigemonam, norcardicin A andtabtoxinine-beta-lactam, a beta-lactamase inhibitor (i.e., clavulanicacid, tazobactam and sulbactam) in conjunction with another beta-lactamantibiotic, an aminoglycoside (i.e., amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, and paromomycin), anansamycin (i.e., geldanamycin and herbimycin), a carbacephem (i.e.,loracarbef), a glycopeptides (i.e., teicoplanin and vancomycin), amacrolide (i.e., azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin andspectinomycin), a monobactam (i.e., aztreonam), a quinolone (i.e.,ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin,sparfloxacin and temafloxacin), a sulfonamide (i.e., mafenide,sulfonamidochrysoidine, sulfacetamide, sulfadiazine, sulfamethizole,sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, trimethoprimand sulfamethoxazole), a tetracycline (i.e., tetracycline,demeclocycline, doxycycline, minocycline and oxytetracycline), anantineoplastic or cytotoxic antibiotic (i.e., doxorubicin, mitoxantrone,bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin,plicamycin, mitomycin, pentostatin and valrubicin) and a miscellaneousantibacterial compound (i.e., bacitracin, colistin and polymyxin B)), anantifungal (i.e., metronidazole, nitazoxanide, tinidazole, chloroquine,iodoquinol and paromomycin), and an antiparasitic (including, but notlimited to, quinine, chloroquine, amodiaquine, pyrimethamine,sulphadoxine, proguanil, mefloquine, atovaquone, primaquine,artemesinin, halofantrine, doxycycline, clindamycin, mebendazole,pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin,rifampin, amphotericin B, melarsoprol, eformithine and albendazole).

For ischemia, a neurological drug may be selected that includes, but isnot limited to, a thrombolytic (i.e., urokinase, alteplase, reteplaseand tenecteplase), a platelet aggregation inhibitor (i.e., aspirin,cilostazol, clopidogrel, prasugrel and dipyridamole), a statin (i.e.,lovastatin, pravastatin, fluvastatin, rosuvastatin, atorvastatin,simvastatin, cerivastatin and pitavastatin), and a compound to improveblood flow or vascular flexibility, including, e.g., blood pressuremedications.

For a behavioral disorder, a neurological drug may be selected from abehavior-modifying compound including, but not limited to, an atypicalantipsychotic (i.e., risperidone, olanzapine, apripiprazole, quetiapine,paliperidone, asenapine, clozapine, iloperidone and ziprasidone), aphenothiazine antipsychotic (i.e., prochlorperazine, chlorpromazine,fluphenazine, perphenazine, trifluoperazine, thioridazine andmesoridazine), a thioxanthene (i.e., thiothixene), a miscellaneousantipsychotic (i.e., pimozide, lithium, molindone, haloperidol andloxapine), a selective serotonin reuptake inhibitor (i.e., citalopram,escitalopram, paroxetine, fluoxetine and sertraline), aserotonin-norepinephrine reuptake inhibitor (i.e., duloxetine,venlafaxine, desvenlafaxine, a tricyclic antidepressant (i.e., doxepin,clomipramine, amoxapine, nortriptyline, amitriptyline, trimipramine,imipramine, protriptyline and desipramine), a tetracyclic antidepressant(i.e., mirtazapine and maprotiline), a phenylpiperazine antidepressant(i.e., trazodone and nefazodone), a monoamine oxidase inhibitor (i.e.,isocarboxazid, phenelzine, selegiline and tranylcypromine), abenzodiazepine (i.e., alprazolam, estazolam, flurazeptam, clonazepam,lorazepam and diazepam), a norepinephrine-dopamine reuptake inhibitor(i.e., bupropion), a CNS stimulant (i.e., phentermine, diethylpropion,methamphetamine, dextroamphetamine, amphetamine, methylphenidate,dexmethylphenidate, lisdexamfetamine, modafinil, pemoline,phendimetrazine, benzphetamine, phendimetrazine, armodafinil,diethylpropion, caffeine, atomoxetine, doxapram, and mazindol), ananxiolytic/sedative/hypnotic (including, but not limited to, abarbiturate (i.e., secobarbital, phenobarbital and mephobarbital), abenzodiazepine (as described above), and a miscellaneousanxiolytic/sedative/hypnotic (i.e. diphenhydramine, sodium oxybate,zaleplon, hydroxyzine, chloral hydrate, aolpidem, buspirone, doxepin,eszopiclone, ramelteon, meprobamate and ethclorvynol)), a secretin (see,e.g., Ratliff-Schaub et al. Autism 9: 256-265 (2005)), an opioid peptide(see, e.g., Cowen et al., J. Neurochem. 89:273-285 (2004)), and aneuropeptide (see, e.g., Hethwa et al. Am. J. Physiol. 289: E301-305(2005)).

For CNS inflammation, a neurological drug may be selected that addressesthe inflammation itself (i.e., a nonsteroidal anti-inflammatory agentsuch as ibuprofen or naproxen), or one which treats the underlying causeof the inflammation (i.e., an anti-viral or anti-cancer agent).

According to one embodiment of the invention, the “coupling” is achievedby generating a multispecific antibody (e.g. a bispecific antibody).Multispecific antibodies are monoclonal antibodies that have bindingspecificities for at least two different sites. In one embodiment, themultispecific antibody comprises a first antigen binding site whichbinds the BBB-R and a second antigen binding site which binds a brainantigen, such as beta-secretase 1 (BACE1) or Abeta, and the other brainantigens disclosed herein.

An exemplary brain antigen bound by such multispecific/bispecificantibody is BACE1, and an exemplary antibody binding thereto is theYW412.8.31 antibody in FIGS. 5 a-b herein.

In another embodiment, the brain antigen is Abeta, exemplary suchantibodies being described in WO2007068412, WO2008011348, WO20080156622,and WO2008156621, expressly incorporated herein by reference, with anexemplary Abeta antibody comprising IgG4 MABT5102A antibody comprisingthe heavy and light chain amino acid sequences in FIGS. 7 a and 7 b,respectively.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (see, e.g. US2006/0025576A1, and Wu et al. Nature Biotechnology (2007)).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to the BBB-R(e.g.TfR) as well as the brain antigen (e.g. BACE1) (see, US2008/0069820, for example).

In one embodiment, the antibody is an antibody fragment, various suchfragments being disclosed above.

In another embodiment, the antibody is an intact or full-lengthantibody. Depending on the amino acid sequence of the constant domain oftheir heavy chains, intact antibodies can be assigned to differentclasses. There are five major classes of intact antibodies: IgA, IgD,IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Theheavy chain constant domains that correspond to the different classes ofantibodies are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known. In one embodiment, the intact antibodylacks effector function.

Techniques for generating antibodies are known and examples providedabove in the definitions section of this document. In one embodiment,the antibody is a chimeric, humanized, or human antibody orantigen-binding fragment thereof.

Various techniques are available for determining binding of the antibodyto the BBB-R. One such assay is an enzyme linked immunosorbent assay(ELISA) for confirming an ability to bind to human BBB-R (and brainantigen). According to this assay, plates coated with antigen (e.g.recombinant sBBB-R) are incubated with a sample comprising theanti-BBB-R antibody and binding of the antibody to the antigen ofinterest is determined.

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

Assays for evaluating uptake of systemically administered antibody andother biological activity of the antibody can be performed as disclosedin the examples or as known for the anti-brain antigen antibody ofinterest.

Exemplary assays where the multispecific antibody binds BACE1 shall nowbe described.

Competition assays may be used to identify an antibody that competeswith any of the antibodies or Fabs descried herein, for example,YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51,Fab12, LC6, LC9, LC10 for binding to BACE1. In certain embodiments, sucha competing antibody binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by any of the antibodies or Fabsdescried herein, for example, YW412.8, YW412.8.31, YW412.8.30,YW412.8.2, YW412.8.29, YW412.8.51, Fab12, LC6, LC9, LC10. Detailedexemplary methods for mapping an epitope to which an antibody binds areprovided in Morris (1996) “Epitope Mapping Protocols,” in Methods inMolecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized BACE1 is incubated in asolution comprising a first labeled antibody that binds to BACE1 (e.g.,YW412.8, YW412.8.31, YW412.8.30, YW412.8.2, YW412.8.29, YW412.8.51,Fab12, LC6, LC9, LC10) and a second unlabeled antibody that is beingtested for its ability to compete with the first antibody for binding toBACE1. The second antibody may be present in a hybridoma supernatant. Asa control, immobilized BACE1 is incubated in a solution comprising thefirst labeled antibody but not the second unlabeled antibody. Afterincubation under conditions permissive for binding of the first antibodyto BACE1, excess unbound antibody is removed, and the amount of labelassociated with immobilized BACE1 is measured. If the amount of labelassociated with immobilized BACE1 is substantially reduced in the testsample relative to the control sample, then that indicates that thesecond antibody is competing with the first antibody for binding toBACE1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14(Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In one aspect, assays are provided for identifying anti-BACE1 antibodiesthereof having biological activity. Biological activity may include,e.g., inhibition of BACE1 aspartyl protease activity. Antibodies havingsuch biological activity in vivo and/or in vitro are also provided, e.g.as evaluated by homogeneous time-resolved fluorescence HTRF assay or amicrofluidic capillary electrophoretic (MCE) assay using syntheticsubstrate peptides, or in vivo in cell lines which express BACE1substrates such as APP.

The antibody (including the multispecific antibody) herein is optionallyrecombinantly produced in a host cell transformed with nucleic acidsequences encoding its heavy and light chains (e.g. where the host cellhas been transformed by one or more vectors with the nucleic acidtherein). The host cell is optionally a mammalian cell, for example aChinese Hamster Ovary (CHO) cell.

III. Pharmaceutical Formulations

Therapeutic formulations of the antibodies used in accordance with thepresent invention are prepared for storage by mixing an antibody havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEENT™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary, optionally those with complementary activities that do notadversely affect each other. The type and effective amounts of suchmedicaments depend, for example, on the amount of antibody present inthe formulation, and clinical parameters of the subjects. Exemplary suchmedicaments are discussed below.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

In one embodiment the formulation is isotonic.

IV. Therapeutic Uses of Anti-BBB-R Antibodies

The anti-BBB-R antibodies (including multispecific antibodies comprisingthem) may be utilized in a variety of in vivo methods. For example, theinvention provides a method of transporting a therapeutic compoundacross the blood-brain barrier comprising exposing the anti-BBB-Rantibody coupled to a therapeutic compound (e.g. a multispecificantibody which binds both the BBB-R and a brain antigen) to the BBB suchthat the antibody transports the therapeutic compound coupled theretoacross the BBB. In another example, the invention provides a method oftransporting a neurological disorder drug across the blood-brain barriercomprising exposing the anti-BBB-R antibody coupled to a brain disorderdrug (e.g. a multispecific antibody which binds both the BBB-R and abrain antigen) to the BBB such that the antibody transports theneurological disorder drug coupled thereto across the BBB. In oneembodiment, the BBB here is in a mammal (e.g. a human), e.g. one whichhas a neurological disorder, including, without limitation: Alzheimer'sdisease (AD), stroke, dementia, muscular dystrophy (MD), multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis,Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick'sdisease, Paget's disease, cancer, traumatic brain injury, etc.

In one embodiment, neurological disorder is selected from: a neuropathy,amyloidosis, cancer (e.g. involving the CNS or brain), an ocular diseaseor disorder, a viral or microbial infection, inflammation (e.g. of theCNS or brain), ischemia, neurodegenerative disease, seizure, behavioraldisorder, lysosomal storage disease, etc.

Neuropathy disorders are diseases or abnormalities of the nervous systemcharacterized by inappropriate or uncontrolled nerve signaling or lackthereof, and include, but are not limited to, chronic pain (includingnociceptive pain), pain caused by an injury to body tissues, includingcancer-related pain, neuropathic pain (pain caused by abnormalities inthe nerves, spinal cord, or brain), and psychogenic pain (entirely ormostly related to a psychological disorder), headache, migraine,neuropathy, and symptoms and syndromes often accompanying suchneuropathy disorders such as vertigo or nausea.

Amyloidoses are a group of diseases and disorders associated withextracellular proteinaceous deposits in the CNS, including, but notlimited to, secondary amyloidosis, age-related amyloidosis, Alzheimer'sDisease (AD), mild cognitive impairment (MCI), Lewy body dementia,Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (Dutchtype); the Guam Parkinson-Dementia complex, cerebral amyloid angiopathy,Huntington's disease, progressive supranuclear palsy, multiplesclerosis; Creutzfeld Jacob disease, Parkinson's disease, transmissiblespongiform encephalopathy, HIV-related dementia, amyotropic lateralsclerosis (ALS), inclusion-body myositis (IBM), and ocular diseasesrelating to beta-amyloid deposition (i.e., macular degeneration,drusen-related optic neuropathy, and cataract).

Cancers of the CNS are characterized by aberrant proliferation of one ormore CNS cell (i.e., a neural cell) and include, but are not limited to,glioma, glioblastoma multiforme, meningioma, astrocytoma, acousticneuroma, chondroma, oligodendroglioma, medulloblastomas, ganglioglioma,Schwannoma, neurofibroma, neuroblastoma, and extradural, intramedullaryor intradural tumors.

Ocular diseases or disorders are diseases or disorders of the eye, whichfor the purposes herein is considered a CNS organ subject to the BBB.Ocular diseases or disorders include, but are not limited to, disordersof sclera, cornea, iris and ciliary body (i.e., scleritis, keratitis,corneal ulcer, corneal abrasion, snow blindness, arc eye, Thygeson'ssuperficial punctate keratopathy, corneal neovascularisation, Fuchs'dystrophy, keratoconus, keratoconjunctivitis sicca, iritis and uveitis),disorders of the lens (i.e., cataract), disorders of choroid and retina(i.e., retinal detachment, retinoschisis, hypertensive retinopathy,diabetic retinopathy, retinopathy, retinopathy of prematurity,age-related macular degeneration, macular degeneration (wet or dry),epiretinal membrane, retinitis pigmentosa and macular edema), glaucoma,floaters, disorders of optic nerve and visual pathways (i.e., Leber'shereditary optic neuropathy and optic disc drusen), disorders of ocularmuscles/binocular movement accommodation/refraction (i.e., strabismus,ophthalmoparesis, progressive external opthalmoplegia, esotropia,exotropia, hypermetropia, myopia, astigmatism, anisometropia, presbyopiaand ophthalmoplegia), visual disturbances and blindness (i.e.,amblyopia, Lever's congenital amaurosis, scotoma, color blindness,achromatopsia, nyctalopia, blindness, river blindness andmicro-opthalmia/coloboma), red eye, Argyll Robertson pupil,keratomycosis, xerophthalmia and andaniridia.

Viral or microbial infections of the CNS include, but are not limitedto, infections by viruses (i.e., influenza, HIV, poliovirus, rubella,),bacteria (i.e., Neisseria sp., Streptococcus sp.,

Pseudomonas sp., Proteus sp., E. coli, S. aureus, Pneumococcus sp.,Meningococcus sp., Haemophilus sp., and Mycobacterium tuberculosis) andother microorganisms such as fungi (i.e., yeast, Cryptococcusneoformans), parasites (i.e., toxoplasma gondii) or amoebas resulting inCNS pathophysiologies including, but not limited to, meningitis,encephalitis, myelitis, vasculitis and abscess, which can be acute orchronic.

Inflammation of the CNS is inflammation that is caused by an injury tothe CNS, which can be a physical injury (i.e., due to accident, surgery,brain trauma, spinal cord injury, concussion) or an injury due to orrelated to one or more other diseases or disorders of the CNS (i.e.,abscess, cancer, viral or microbial infection).

Ischemia of the CNS, as used herein, refers to a group of disordersrelating to aberrant blood flow or vascular behavior in the brain or thecauses therefor, and includes, but is not limited to: focal brainischemia, global brain ischemia, stroke (i.e., subarachnoid hemorrhageand intracerebral hemorrhage), and aneurysm.

Neurodegenerative diseases are a group of diseases and disordersassociated with neural cell loss of function or death in the CNS, andinclude, but are not limited to: adrenoleukodystrophy, Alexander'sdisease, Alper's disease, amyotrophic lateral sclerosis, ataxiatelangiectasia, Batten disease, cockayne syndrome, corticobasaldegeneration, degeneration caused by or associated with an amyloidosis,Friedreich's ataxia, frontotemporal lobar degeneration, Kennedy'sdisease, multiple system atrophy, multiple sclerosis, primary lateralsclerosis, progressive supranuclear palsy, spinal muscular atrophy,transverse myelitis, Refsum's disease, and spinocerebellar ataxia.

Seizure diseases and disorders of the CNS involve inappropriate and/orabnormal electrical conduction in the CNS, and include, but are notlimited to: epilepsy (i.e., absence seizures, atonic seizures, benignRolandic epilepsy, childhood absence, clonic seizures, complex partialseizures, frontal lobe epilepsy, febrile seizures, infantile spasms,juvenile myoclonic epilepsy, juvenile absence epilepsy, Lennox-Gastautsyndrome, Landau-Kleffner Syndrome, Dravet's syndrome, Otahara syndrome,West syndrome, myoclonic seizures, mitochondrial disorders, progressivemyoclonic epilepsies, psychogenic seizures, reflex epilepsy, Rasmussen'sSyndrome, simple partial seizures, secondarily generalized seizures,temporal lobe epilepsy, toniclonic seizures, tonic seizures, psychomotorseizures, limbic epilepsy, partial-onset seizures, generalized-onsetseizures, status epilepticus, abdominal epilepsy, akinetic seizures,autonomic seizures, massive bilateral myoclonus, catamenial epilepsy,drop seizures, emotional seizures, focal seizures, gelastic seizures,Jacksonian March, Lafora Disease, motor seizures, multifocal seizures,nocturnal seizures, photosensitive seizure, pseudo seizures, sensoryseizures, subtle seizures, sylvan seizures, withdrawal seizures, andvisual reflex seizures).

Behavioral disorders are disorders of the CNS characterized by aberrantbehavior on the part of the afflicted subject and include, but are notlimited to: sleep disorders (i.e., insomnia, parasomnias, night terrors,circadian rhythm sleep disorders, and narcolepsy), mood disorders (i.e.,depression, suicidal depression, anxiety, chronic affective disorders,phobias, panic attacks, obsessive-compulsive disorder, attention deficithyperactivity disorder (ADHD), attention deficit disorder (ADD), chronicfatigue syndrome, agoraphobia, post-traumatic stress disorder, bipolardisorder), eating disorders (i.e., anorexia or bulimia), psychoses,developmental behavioral disorders (i.e., autism, Rett's syndrome,Aspberger's syndrome), personality disorders and psychotic disorders(i.e., schizophrenia, delusional disorder, and the like).

Lysosomal storage disorders are metabolic disorders which are in somecases associated with the CNS or have CNS-specific symptoms; suchdisorders include, but are not limited to: Tay-Sachs disease, Gaucher'sdisease, Fabry disease, mucopolysaccharidosis (types I, II, III, IV, V,VI and VII), glycogen storage disease, GM1-gangliosidosis, metachromaticleukodystrophy, Farber's disease, Canavan's leukodystrophy, and neuronalceroid lipofuscinoses types 1 and 2, Niemann-Pick disease, Pompedisease, and Krabbe's disease.

In one aspect, the antibody is used to detect a neurological disorderbefore the onset of symptoms and/or to assess the severity or durationof the disease or disorder. In one aspect, the antibody permitsdetection and/or imaging of the neurological disorder, including imagingby radiography, tomography, or magnetic resonance imaging (MRI).

In one aspect, a low affinity anti-BBB-R antibody for use as amedicament is provided. In further aspects, a low affinity anti-BBB-Rantibody for use in treating a neurological disease or disorder isprovided (e.g., Alzheimer's disease). In certain embodiments, a lowaffinity anti-BBB-R antibody for use in a method of treatment isprovided. In certain embodiments, the invention provides a low affinityanti-BBB-R antibody for use in a method of treating an individual havinga neurological disease or disorder comprising administering to theindividual an effective amount of the anti-BBB-R antibody (optionallycoupled to a neurological disorder drug). In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent. In furtherembodiments, the invention provides an anti-BBB-R antibody for use inreducing or inhibiting amlyoid plaque formation in a patient at risk orsuffering from a neurological disease or disorder (e.g., Alzheimer'sdisease). An “individual” according to any of the above embodiments isoptionally a human. In certain aspect, the anti-BBB-R antibody for usein the methods of the invention improves uptake of the neurologicaldisorder drug with which it is coupled.

In a further aspect, the invention provides for the use of a lowaffinity anti-BBB-R antibody in the manufacture or preparation of amedicament. In one embodiment, the medicament is for treatment ofneurological disease or disorder. In a further embodiment, themedicament is for use in a method of treating neurological disease ordisorder comprising administering to an individual having neurologicaldisease or disorder an effective amount of the medicament. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent.

In a further aspect, the invention provides a method for treatingAlzheimer's disease. In one embodiment, the method comprisesadministering to an individual having Alzheimer's disease an effectiveamount of a multispecific antibody which binds both BACE1 and TfR. Inone such embodiment, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent. An “individual” according to any of the above embodiments may bea human.

The anti-BBB-R antibodies of the invention can be used either alone orin combination with other agents in a therapy. For instance, theanti-BBB-R antibody of the invention may be co-administered with atleast one additional therapeutic agent. In certain embodiments, anadditional therapeutic agent is a therapeutic agent effective to treatthe same or a different neurological disorder as the anti-BBB-R antibodyis being employed to treat. Exemplary additional therapeutic agentsinclude, but are not limited to: the various neurological drugsdescribed above, cholinesterase inhibitors (such as donepezil,galantamine, rovastigmine, and tacrine), NMDA receptor antagonists (suchas memantine), amyloid beta peptide aggregation inhibitors,antioxidants, γ-secretase modulators, nerve growth factor (NGF) mimicsor NGF gene therapy, PPARγ agonists, HMS-CoA reductase inhibitors(statins), ampakines, calcium channel blockers, GABA receptorantagonists, glycogen synthase kinase inhibitors, intravenousimmunoglobulin, muscarinic receptor agonists, nicrotinic receptormodulators, active or passive amyloid beta peptide immunization,phosphodiesterase inhibitors, serotonin receptor antagonists andanti-amyloid beta peptide antibodies. In certain embodiments, the atleast one additional therapeutic agent is selected for its ability tomitigate one or more side effects of the neurological drug.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with other interventional therapies such as, butnot limited to, radiation therapy, behavioral therapy, or othertherapies known in the art and appropriate for the neurological disorderto be treated or prevented.

The anti-BBB-R antibody of the invention (and any additional therapeuticagent) can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Lipid-based methods of transporting the antibody or fragment thereofacross the blood-brain barrier include, but are not limited to,encapsulating the antibody or fragment thereof in liposomes that arecoupled to antibody binding fragments that bind to receptors on thevascular endothelium of the blood-brain barrier (see e.g., U.S. PatentApplication Publication No. 20020025313), and coating the antibody oractive fragment thereof in low-density lipoprotein particles (see e.g.,U.S. Patent Application Publication No. 20040204354) or apolipoprotein E(see e.g., U.S. Patent Application Publication No. 20040131692).

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an immunoconjugate of the invention inplace of or in addition to an anti-BBB-R antibody.

V. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an immunoconjugate of the invention in place of or in additionto an anti-BBB-R antibody.

The article of manufacture optionally further comprises a package insertwith instructions for treating a neurological disorder in a subject,wherein the instructions indicate that treatment with the antibody asdisclosed herein treats the neurological disorder, and optionallyindicates that the antibody has improved uptake across the BBB due toits low affinity for the BBB-R.

III. Examples

This example evaluated the transferrin receptor (TfR), which mediatesiron transport into the brain via the holo-transferrin complex(Skarlatos et al. Brain Res 683: 164-171 (1995)). A human chimericanti-murine transferrin receptor (anti-TfR^(A)) antibody that does notcompete with endogenous transferrin binding to TfR was compared to ahuman control IgG in a double-labeled experiment for uptake in wild typemouse brain. A single trace dose (approximately 50 μg/kg) of[¹³¹I]anti-TfR^(A) and [¹²⁵I]control IgG was injected into wild typemice intravenously (i.v.) and brain uptake was measured at 5 min., 0.5,1, 4, 24, 48, and 72 hours. A significant increase in [¹³¹I]anti-TfR^(A)uptake in the brain, measured as a percentage of injected dose per gramof brain, was observed at all time points (FIG. 1A). At its peak, 1 hourafter injection, there was a >11-fold difference in [¹³¹I]anti-TfR^(A)brain accumulation as compared to [¹²⁵I]control IgG (n=6). If unlabeledanti-TfR^(A) (4 mg/kg body weight) was also co-administered, brainaccumulation of [¹³¹I]anti-TfR^(A) was nearly reduced to the level ofcontrol IgG, indicating specific, target-driven uptake.

To evaluate whether significant antibody uptake in brain also occurs attherapeutic dose levels, wild type mice were administered eitheranti-TfR^(A) or control IgG at 20 mg/kg intravenously (i.v.). Humanantibody concentration in the cortex and serum was determined 1 and 24hours after injection using a human Fc sandwich ELISA. Briefly, afterthe indicated time of administration, mice were perfused with D-PBS at arate of 2 ml/min. for 8 minutes. Brains were extracted and the cortexand hippocampus were isolated, homogenized in 1% NP-40 (Cal-Biochem) inPBS containing Complete Mini EDTA-free protease inhibitor cocktailtablets (Roche Diagnostics). Homogenized brain samples were rotated at4° C. for 1 hour before centrifugation at 14,000 rpm for 20 minutes. Thesupernatant was isolated for brain antibody measurement. Whole blood wascollected prior to perfusion in serum separator microcontainer tubes (BDDiagnostics), allowed to clot for at least 30 minutes, and spun down at5,000×g for 90 seconds. The supernatant was isolated for serum antibodymeasurements. Antibody concentrations in mouse serum and brain sampleswere measured by ELISA. NUNC 384-well Maxisorp immunoplates (Neptune,N.J.) were coated with F(ab′)2 fragment of donkey anti-human IgG, Fcfragment-specific polyclonal antibody (Jackson ImmunoResearch, WestGrove, Pa.) overnight at 4° C. Plates were blocked with PBS containing0.5% BSA for 1 hour at 25° C. Each antibody was used as an internalstandard to quantify respective antibody concentration. Plates werewashed with PBS containing 0.05% Tween-20 using a microplate washer(Bio-Tek Instruments, Inc., Winooski, Vt.). Standards and samples werediluted in PBS containing 0:5% BSA, 0.35 M NaCl, 0.25% CHAPS, 5 mM EDTA,0.05% Tween-20 and 15 ppm Proclin, and were added to the microplate fortwo hours at 25° C. Bound antibody was detected with horseradishperoxidase-conjugated F(ab′)2 goat anti-human IgG, Fc-specificpolyclonal antibody (Jackson ImmunoResearch), developed using3,3′,5,5′-tetramethyl benzidine (TMB) (KPL, Inc., Gaithersburg, Md.) andabsorbance measured at 450 nm on a Multiskan Ascent reader (ThermoScientific, Hudson, N.H.). Concentrations were determined from thestandard curve using a four-parameter non-linear regression program. Theassay had lower limit of quantification (LLOQ) values of 3.12 ng/ml inserum and 15.6 ng/g in brain. Statistical analysis of differencesbetween experimental groups was performed using a two-tailed unpairedt-test.

Compared to control IgG, concentration of anti-TfR^(A) was significantlyhigher in the brain 24 hours after antibody administration (FIG. 1B,p=0.0002, n=10). Additionally, human IgG concentration in brainwas >2.5-fold higher compared to serum for anti-TfR^(A) compared tocontrol IgG at 24 hours (FIG. 1C, p=0.003, n=10). Together with theradiolabeled trace data, these results indicate that systemicallyadministered anti-TfR^(A) can accumulate in the brain, however thetissue distribution of antibody in brain remained to be understood.

To address the distribution of systemically administered antibodies inbrain, wild type mice were injected 20 mg/kg i.v. with eitheranti-TfR^(A) or control IgG, perfused with PBS to flush out anyremaining circulating antibody, and brain sections were stained withfluorescent anti-human secondary IgG to determine antibody localization.After 1 hour of circulation, anti-TfR^(A) had a pronounced vasculardistribution, as indicated by its co-localization with the basementmembrane marker anti-collagen IV (FIG. 1D, left column). Although lesspronounced, control IgG also localized to the vasculature, indicatingthat after 1 hour of exposure, therapeutic dose levels of systemicallyadministered IgG maintains a vascular distribution (FIG. 1E, leftcolumn). However, there was a marked difference in antibody localization24 hours after injection. Anti-TfR^(A) distribution was no longerexclusively vascular, but instead, exhibited modest parenchymal staining(FIG. 1D, right columns). In contrast, control IgG antibody was largelyabsent in brain tissue 24 hours after injection (FIG. 1E, rightcolumns). These results indicate that when dosed at therapeuticallyrelevant levels, anti-TfR^(A) may penetrate the BBB as evidenced bymodest parenchymal staining, however, the bulk of the brain-accumulatedantibody was largely confined to endothelial cells of the BBB.

Accumulation in the parenchyma requires binding to surface TfRsexpressed on brain endothelial cells as well as dissociation from thereceptor following RMT. Without being bound by any theory, it washypothesized that reduced affinity for TfR may facilitate dissociationafter RMT and allow enhanced accumulation in the parenchyma. Further, ananti-TfR with reduced affinity would be less efficiently captured andtransported in a concentration-limited environment, such as in thebrain, where anti-TfR concentrations are low. In a clinical setting,however, serum levels of an anti-TfR therapeutic would still besufficiently high to maintain saturation of the receptor in the vascularlumen.

To test this prediction, variants of anti-TfR^(A) were generated thatvary in their binding affinity for TfR. These variants were tested in acompetition ELISA assay (FIG. 2A); anti-TfR^(A) had the strongestaffinity and lowest IC50 of any of the tested antibodies for TfR, andeach of anti-TfR^(B,C,D) had successively lower affinity and higherIC50. Later, variant anti-TfR^(E) was generated and tested in the sameassay along with anti-TfR^(A,B,C,D) variants; as shown in FIG. 2A, itwas substantially less able to compete for binding to TfR than any ofthe other tested anti-TfR antibodies, and it had a corresponding highIC50 value (Table 2).

TABLE 2 IC50 measurements for anti-TfR antibodies Antibody IC50 (nM)Standard deviation Anti-TfR^(A) 1.7 0.1 Anti-TfR^(B) 6.9 0.4Anti-TfR^(C) 65 12 Anti-TfR^(D) 111 16 Anti-TfR^(E) >5 × 10⁴ —

These variants were tested in both a non-TfR saturating (trace dosing)and TfR saturating (therapeutic dosing) environment. Trace levels of[¹²⁵I]anti-TfR^(A), [¹²⁵I]anti-TfR^(B), [¹²⁵I]anti-TfR^(C),[¹²⁵I]anti-TfR^(D) and [¹²⁵I]anti-TfR^(E) (which vary in affinity forTfR, with the affinity of anti-TfR^(A)>affinity of anti-TfR^(B)>affinityof anti-TfR^(C)>affinity of anti-TfR^(D)>affinity of anti-TfR^(E)) wereinjected i.v. into mice and brain uptake was measured 1, 4, or 24 hoursafter injection. This assay was performed originally with[¹²⁵I]anti-TfR^(A), [¹²⁵I]anti-TfR^(B), [¹²⁵I]anti-TfR^(C), and[¹²⁵I]anti-TfR^(D), and later repeated upon the construction of[¹²⁵I]anti-TfR^(E), the results of which are shown in FIG. 2B.Consistent with the proposed model, trace dose levels of lower affinityanti-TfR antibodies resulted in less uptake in brain compared to higheraffinity variants (FIG. 2B). In striking contrast to trace dosing,however, brain uptake of these same lower-affinity variants attherapeutic levels (20 mg/kg assessed at 1 and 24 hours) exhibitedincreased brain uptake at 24 hours as affinity was lowered, while nosignificant difference in uptake was observed at 1 hour (FIG. 2C). Thesedata support the hypothesis that a lower affinity RMT antibody wouldexhibit decreased transport under limiting concentrations whiletransport under saturating conditions would be unaffected.

Thus, the following model is proposed: compared to a high affinityantibody, fewer low affinity antibodies bind to receptors on the luminalside of the vasculature under non-saturating concentrations, leading tolower endothelial uptake (FIG. 2D, left panels). At a higher therapeuticdose, however, luminal receptors would be saturated regardless ofaffinity resulting in similar endothelial uptake (FIG. 2D, rightpanels). Under these conditions, lower affinity RMT antibodies canachieve greater brain accumulation by 1) maximizing dissociation fromthe RMT target facilitating release into the brain, and 2) reducing thelikelihood of efflux out of the brain as concentrations are limited onthe parenchymal side of the BBB. Thus in a therapeutic setting, a loweraffinity antibody for an RMT target is surprisingly advantageous forparenchymal accumulation.

To evaluate the localization of these variants exhibiting increasedbrain uptake, mice were dosed i.v. with 20 mg/kg of either the highaffinity anti-TfR^(A) or the lower affinity variants anti-TfR^(B,C,D).After 24 hours, animals were PBS-perfused and brain sections wereco-stained for human IgG and the neuronal marker NeuN (FIG. 2E). Asobserved before, high affinity anti-TfR^(A) treated animals had mostlyvascular staining with low levels of parenchymal signal (FIG. 2E, toprow). However, the lower affinity anti-TfR^(B,C,D) had noticeably morepronounced cellular staining not depicting cortical blood vessels (FIG.2E, data for anti-TfR^(B,C,D)). Furthermore, co-localized staining withNeuN indicated a redistribution of antibody from the vasculature toneurons. This is especially pronounced in a representative highermagnification image of anti-TfR^(D) variant (FIG. 2F). Together with thebrain uptake data, these results indicate that a significantly higherbrain accumulation of antibody can be achieved by lowering the affinityof anti-TfR for TfR, and that lower affinity antibodies such asanti-TfR^(D) selectively distributed to neurons.

Transport of anti-TfR antibodies across the BBB was further establishedwhen evaluating a bispecific antibody (anti-TfR^(A)/BACE1) that bindsboth TfR and the amyloid precursor protein (APP) cleavage enzyme, betasecretase (BACE1) (FIG. 3A). The high affinity anti-TfR^(A) was used toengineer the TfR binding arm of the bispecific using standard ‘knob inhole’ bispecific antibody construction technology (see, e.g., Ridgway etal., Protein Eng. (1996) 9(7): 617-621). In addition to the knob andhole mutations in the Fc for anti-TfR (hole) and anti-BACE1 (knob), theanti-TfR arm of the antibody comprised a mutation in the Fc region thatabrogated glycosylation (N297G). The knob and hole half-antibodies werepurified separately and annealed to generate an aglycosylated bispecificIgG in vitro. The binding affinity of the anti-TfR^(A)/BACE1 antibody toTfR was considerably reduced compared to the parental anti-TfR^(A) dueto the loss of bivalent binding (FIG. 3B). BACE1 is expressed primarilyon neurons in the CNS and is considered to be the primary contributor ofbeta amyloid (Aβ₁₋₄₀) formation via APP cleavage (Vassar et al., Science286:735-741 (1999)). An antibody to BACE1 has been described as aneffective means to inhibit BACE1 activity, and may reduce Aβ₁₋₄₀production in vivo. Inhibition of BACE1 by anti-TfR/BACE1 was examinedin a HEK293 cell line stably expressing APP. Compared to anti-BACE1, thebispecific antibody had both similar efficacy and potency in inhibitingAβ₁₋₄₀ production, suggesting that the anti-TfR/BACE1 is a fullyfunctional large molecule inhibitor of BACE1 activity (FIG. 3C).

Based on this model, this lower affinity bispecific would be expected tobe a more favorable candidate for increased uptake compared to theanti-TfR alone. To investigate the brain accumulation of the bispecificantibody, trace doses of [¹²⁵I]anti-TfR^(A)/BACE1 were compared to[¹²⁵I]anti-TfR^(A) and [¹²⁵I]anti-BACE1 and brain uptake was evaluatedat 30 min., 6, 24, and 48 hours after i.v. injection. Significantlyhigher brain uptake was observed with [¹²⁵I]anti-TfR/BACE1 compared to[¹²⁵I]anti-BACE1 at all time points (FIG. 3 d, n=4). Consistent with theaffinity hypothesis, brain uptake of this non-saturating trace dose of[¹²⁵I]anti-TfR^(A) was much greater than that of the lower affinity[¹²⁵I]anti-TfR^(A)/BACE1. To assess antibody accumulation at therapeuticdose levels, mice were injected i.v. with anti-TfR^(A)/BACE1 oranti-BACE1 at 20 mg/kg and brain uptake of, antibody was determinedafter 1, 12, 24, and 48 hours. Compared to the monospecific anti-BACE1,administration of the bispecific anti-TfR^(A)/BACE1 resulted in asignificantly higher brain uptake at all time points (FIG. 3E). Aspredicted by the affinity model, the extent of uptake was significantlygreater than the higher affinity anti-TfR^(A) alone (compare FIG. 3E to1B). Peak accumulation was achieved at 24 hours after injection,reaching concentrations of ˜20 nM and remained elevated 48 hours afterinjection, even as peripheral levels of antibody cleared to ˜12% of itsconcentration at 1 hour. Enhanced uptake by the bispecific isdramatically apparent when comparing the average percent of antibody inthe brain versus the serum (FIG. 3F).

To determine localization of anti-TfR^(A)/BACE1 after systemicadministration, mice were PBS-perfused 24 hours after injection, andantibody distribution was visualized with anti-human fluorescentsecondary (FIG. 3G). Similar to the lower affinity anti-TfR antibodylocalization, there was substantial staining of the parenchyma inaddition to vascular staining. Parenchymal co-localization with NeuNindicated that these antibodies were localized to the neuronalpopulation.

In contrast, animals injected with control IgG showed a complete lack ofboth vascular and parenchymal staining. Together, these data indicatethat the bispecific anti-TfR^(A)/BACE1 traverses across the BBB and cansignificantly accumulate in the brain parenchyma.

To assess the efficacy of anti-TfR^(A)/BACE1 on Aβ₁₋₄₀ production invivo, wild type mice were administered a single 25 mg/kg or 50 mg/kgdose of control IgG, anti-BACE1, or anti-TfR/BACE1. Based on theobservation that brain antibody uptake peaks 24 hours after injection(see FIG. 3E), brain and plasma Aβ₁₋₄₀ levels were determined at 24 and48 hours after i.v. antibody administration. At 25 mg/kg,anti-TfR^(A)/BACE1 was able to significantly reduce brain Aβ₁₋₄₀ levelscompared to control IgG both after 24 (p=0.001, n=10) and 48 (p=0.0003,n=10) hours post-injection, while anti-BACE1 had no effect on Aβ₁₋₄₀reduction (FIG. 4A). At 50 mg/kg, anti-TfR^(A)/BACE1 had a even moredramatic effect on reducing brain Aβ₁₋₄₀ at both time points compared tocontrol IgG (FIG. 3B, p<0.0001, n=10 for both 24 and 48 hr).Administration of anti-BACE1 at this dose also significantly reducedbrain Aβ₁₋₄₀ levels compared to control (p<0.0001, n=10 for 24 hr;p=0.006, n=10 for 48 hr), though to a significantly lesser extent thanthe bispecific anti-TfR^(A)/BACE1 (p<0.0001, n=10 for both 24 and 48hours). Notably, the ability of the bispecific to reduce Aβ₁₋₄₀ was 2-to 3-fold greater than anti-BACE1 for all time points and dosesmeasured. The maximal effect of anti-TfR^(A)/BACE1 was achieved 48 hoursafter injection at 50 mg/kg, with a 50.0±1.9% reduction in brain Aβ₁₋₄₀compared to control IgG (FIG. 4E). Significant reductions in peripheralAβ₁₋₄₀ was also observed at both doses and time points foranti-TfR^(A)/BACE1 (FIG. 4C-D). Treatment with anti-BACE1 resulted in areduction in peripheral Aβ₁₋₄₀ only at the 24 hour time point (p=0.01for 25 mg/kg, p=0.002 for 50 mg/kg; n=10 for each). These data confirmthat antibodies engineered to cross the BBB can be pharmacodynamicallyefficacious. Furthermore, the increase in brain penetrance of thebispecific renders it more potent BACE1 inhibitor drug by significantlyreducing brain Aβ₁₋₄₀ levels.

At therapeutic doses of 20 mg/kg, however, BBB penetrance and entry intothe non-vascular portions of the CNS was enhanced in theanti-TfR^(E)-treated animals relative to the anti-TfR^(A) oranti-TfR^(D)-treated animals (FIG. 8B). Notably, anti-TfR^(D) achieved ahigher initial concentration in the brain, which steadily decreasedafter day 2; anti-TfR^(E), on the other hand, retained a consistentlyhigh level of brain exposure over the tested 8 day period. Relatedly,anti-TfR^(E) concentration in the serum decreased the least of all ofthe anti-TfR antibodies over the assessed period (FIG. 8A). In all, thisdata indicates that generally a lower affinity for TfR surprisinglyreduces serum clearance and increases brain exposure, but that at somethreshold the lower affinity begins to impair the maximum brain exposureobtainable with the antibody. In this example, an optimum would seem tobe found between the affinities of antibodies anti-TfR^(D) andanti-TfR^(E) for transferrin receptor.

Importantly, these data highlight several causative mechanisms behindincreasing uptake of an antibody into the CNS using a lower-affinityantibody approach. First, high affinity anti-TfR antibodies (e.g.,anti-TfR^(A), FIG. 1D) limit brain uptake by quickly saturating the TfRin the brain vasculature, thus reducing the total amount of antibodytaken up into the brain and also restricting its distribution to thevasculature. Strikingly, lowering affinity (e.g., anti-TfR^(B-E), andanti-TfR^(A,D,E)/BACE1, FIG. 2C, 2E, 2F, 3E-G and 9C) improves brainuptake and distribution, with a robust shift observed in localizationfrom the vasculature to neurons and associated neuropil. Second, thelower affinity of the antibody for TfR is proposed to impair the abilityof the antibody to return to the vascular side of the BBB via TfR fromthe CNS side of the membrane because the overall affinity of theantibody for TfR is low and the local concentration of the antibody onthe CNS side of the BBB is non-saturating due to the rapid dispersal ofthe antibody into the CNS compartment (see, e.g., FIGS. 1D, 2E and 2F).Third, in vivo, antibodies with less affinity for TfR are not clearedfrom the system as efficiently as those with greater affinity for TfR(see FIGS. 8A and 9B), and thus remain at higher circulatingconcentrations than their higher-affinity counterparts. This isadvantageous because the circulating antibody levels of thelower-affinity antibody are sustained at therapeutic levels for a longerperiod of time than the higher-affinity antibody, which subsequentlyimproves uptake of antibody in brain for longer period of time (compareanti-TfR^(A)/BACE1 to anti-TfR^(D)/BACE1 in FIG. 9C). Furthermore, thisimprovement in both plasma and brain exposure may reduce the frequencyof dosing in the clinic, which would have potential benefit not only forpatient compliance and convenience but also in lessening any potentialside effects or off-target effects of the antibody and/or of atherapeutic compound coupled thereto.

Further studies were performed to assess whether further lessening theaffinity of the bispecific anti-TfR^(A)/BACE1 antibody could furtherimprove its BBB and parenchymal penetrance. Two further bispecificantibodies were constructed: anti-TfR^(D)/BACE1 and anti-TfR^(E)/BACE1,using the same construction methodology employed for theanti-TfR^(A)/BACE1 antibody. Competition ELISA assays were performed(FIG. 9A) and the resulting IC50s were as follows:

TABLE 3 IC50/affinity measurements for anti-TfR/BACE1 antibodiesAntibody IC50 Kd (Biacore) (nM) Anti-TfR^(A)/BACE1 15 nM 33.3 ± 1.7Anti-TfR^(D)/BACE1 1.6 μM 630 ± 50 Anti-TfR^(E)/BACE1 >50 μM N.D.

Surface plasmon resonance measurements of Kd were also made for thebinding between each bispecific antibody and TfR. The BIACORE® analysisin Table 3 was performed as follows. Kd was measured using surfaceplasmon resonance assays using a BIACORE®-T-100 (BIAcore, Inc.,Piscataway, N.J.) at 25° C. using penta-His Ab capture (Qiagen,Valencia, Calif.). Briefly, carboxymethylated dextran biosensor chips(CM5, BIACORE, Inc.) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Penta-His antibody was diluted with 100 mM sodium acetate, pH 4.0, to 50μg/ml before injection at a flow rate of 5 μl/minute to achieveapproximately 10000 response units (RU) of coupled protein. Followingthe injection of antibody, 1 M ethanolamine was injected to blockunreacted groups. For kinetics measurements, MuTfR-H is was injected inHBS-P to reach about 50 RU, then two-fold serial dilutions ofanti-TfR^(A)/BACE1 (1.95 nM to 1000 nM) or anti-TfR^(D)/BACE1 (9.75 nMto 5000 nM) were injected in HBS-P at 25° C. at a flow rate ofapproximately 30 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) were calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) was calculated as the ratiokoff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). Theaffinity of anti-TfR^(E)/BACE1 was too weak to be determined by surfaceplasmon resonance.

As shown in FIG. 9A and Tables 2 and 3, the bispecificanti-TfR^(A)/BACE1 and anti-TfR^(D)/BACE1 antibodies bound markedly lesswell to TfR than the corresponding monospecific anti-TfR^(A) andanti-TfR^(D) (compare FIG. 9A to FIG. 2A). For anti-TfR^(E)/BACE1 thepartial binding curves for the bispecific and monospecific also suggestthat the bispecific anti-TfR^(E)/BACE1 has a much higher IC50 than thecorresponding monospecific anti-TfR^(E).

These antibodies were tested in the same in vivo Aβ₁₋₄₀ production assaydescribed above. Briefly, 6-8 week old wild type C57B1/6 mice wereadministered via i.v. tail vein injection a single 50 mg/kg dose ofcontrol IgG, monospecific anti-BACE1, or anti-TfR^(A)/BACE1anti-TfR^(D)/BACE1, or anti-TfR^(E)/BACE1, 6 mice per antibody treatmentper timepoint, for a total of 180 mice treated. Brain and plasma Aβ₁₋₄₀levels were determined at 1, 2, 4, 6, 8, and 10 days after i.v. antibodyadministration (FIGS. 9B-9E). The concentration of bispecific antibodiesfound in the brain (FIG. 9C) at the earliest time point was greatestwith anti-TfR^(A)/BACE1 and anti-TfR^(D)/BACE1, each of which hadconcentrations of more than twice the concentration achieved byanti-TfR^(E)/BACE1 at the 1 day time point. However, theanti-TfR^(A)/BACE1 brain concentration levels returned to control levelsby day 6, and the anti-TfR^(D)/BACE1 levels did so by day 10. Incontrast, the lowest affinity bispecific antibody anti-TfR^(E)/BACE1 hada much lower relative dropoff in brain antibody concentration ascompared to anti-TfR^(A)/BACE1 and anti-TfR^(D)/BACE1, in keeping withthe proposed model that a lower affinity for anti-TfR leads to a reducedability for the antibody to be exported from the parenchymal space. Thelevels of Abeta1-40 in the brain (FIG. 9E) were reduced in roughly thesame proportion expected by the observed concentrations of bispecificantibody in the brain: anti-TfR^(A)/BACE1 and anti-TfR^(D)/BACE1 hadsimilarly reduced levels of observed brain Abeta1-40 at the earliesttime points (days 1-2), which either rapidly increased at subsequenttime points (anti-TfR^(A)/BACE1) or more moderately increased atsubsequent time points (anti-TfR^(D)/BACE1), consistent with theobserved decreases in brain concentration of each of these antibodies(FIG. 9C). Notably, while the anti-TfR^(E)/BACE1 antibody treatmentresulted in a relatively more modest reduction in brain Abeta1-40 levelsthan that observed with the other bispecific antibodies, this reductionwas consistent across all timepoints (FIG. 9E).

Plasma measurements (FIG. 9B) showed that anti-TfR^(A)/BACE1 was clearedby day 4, while anti-TfR^(D)/BACE1 persisted at relatively low levelsacross all time points, and levels of anti-TfR^(E)/BACE1 remainedsimilar to controls across all time points. Consistent with thisfinding, observed plasma Abeta1-40 levels (FIG. 9D) were similarlyreduced from control anti-gD levels at all time points with each ofanti-TfR^(D)/BACE1, anti-TfR^(E)/BACE1 and anti-BACE1.Anti-TfR^(A)/BACE1 showed similar reductions at the 1, 2 and 4-day timepoints, rapidly returning to control levels at later time points, inkeeping with the observed disappearance of the antibody from the plasma.

These results again demonstrate that bispecific anti-TfR/BACE1antibodies effectively cross the BBB and inhibit BACE1 activity in amammalian in vivo system. They also suggest that an affinity for TfRbetween that of the anti-TFR^(D)/BACE1 and the anti-TfR^(E)/BACE1 mayprovide an optimal combination of persistence and activity in theparenchyma/brain. It is noted, however that for each brain target, thepotency of the bispecific arm specific for that target will dictate howmuch of the anti-TfR/target bispecific antibody must be present in theCNS side of the BBB in order to achieve the desired results, and thuswhat degree of affinity of the anti-TfR for TfR must be used in thebispecific to obtain that concentration. This invention provides a meansto determine and design the bispecific antibody to achieve such targetlevels in the CNS after administration on the nonprivileged side of theBBB. These results were confirmed and extended using another anti-TfRbispecific antibody, anti-TfR/Abeta, that binds both TfR and amyloidbeta. Three bispecific variants were prepared: anti-TfR^(A)/Abeta,anti-TfR^(D)/Abeta and anti-TfR^(E)/Abeta, using the same methods asused for the preparation of the anti-TfR/BACE1 bispecific above. Abetais the main substituent of amyloid plaques, which are believed to beinvolved in the development of AD. Inhibition of plaque formation byAbeta binding and removal, in either its free or oligomerized state, mayinhibit the development or progression of AD. The pharmacokineticproperties of each of these bispecific antibodies were assessed.

A single 50 mg/kg dose of control IgG, monospecific anti-Abeta antibody,or each bispecific antibody was injected i.p. into 8-16 week old wildtype C57BL/6J mice or mice expressing both human presenilin 2 and humanamyloid precursor protein (PS2APP). Due to a limited number oftransgenic animals, only two of the bispecific variants(anti-TfR^(D)/Abeta and anti-TfR^(E)/Abeta) were assayed in the PS2APPmice. Four to six replicate mice were dosed in each treatment group. Themice were sacrificed after 24 hours and drug levels measured in both thebrain and plasma, as with the anti-TfR/BACE1 bispecific studies. Priorto sacrifice, blood was also collected 6 hours post-dose for an earlyevaluation of plasma antibody concentrations. Plasma measurements ofantibody concentration (FIGS. 10A and 11A) showed that all antibodieswere present at similar levels at 6 hours post-dose. However, controlmonospecific anti-Abeta levels were reduced compared to control IgG by24 hours. This is similar to previous observations for this anti-Abetamolecule. At 24 hours, anti-TfR^(A)/Abeta showed similar antibody levelsin the periphery as anti-Abeta, whereas anti-TfR^(D)/Abeta andanti-TfR^(E)/Abeta showed slightly elevated levels, intermediate betweenanti-Abeta and control IgG.

The concentration of bispecific antibodies found in the brain wasincreased compared to both control IgG and anti-Abeta (FIGS. 10B and11B). As compared to anti-Abeta, anti-TfR^(A)/Abeta had concentrations12-fold higher, anti-TfR^(D)/Abeta had concentrations 8- to 15-foldhigher, and anti-TfR^(E)/Abeta had concentrations 4- to 5-fold higher.The increase in brain uptake of the anti-TfR^(A,D,E)/Abeta antibodiescompared to anti-Abeta was even greater than the increases seen for theanti-TfR^(A,D,E)/BACE bispecific antibodies compared to anti-BACE1. Thisis likely due to the decreased peripheral exposure of anti-Abetacompared to control IgG 24 hours after dosing, which resulted in lowerlevels of anti-Abeta in brain as compared to control IgG.

These findings are the first demonstration that large moleculeantibodies administered at therapeutically relevant doses can traversethe BBB and produce significant and sustained brain uptake. Furthermore,these results demonstrating an inverse relationship between antibodyaffinity and extent of brain uptake furthers understanding of RMTdynamics. This novel insight can be applied to a variety of otherpotential RMT targets to provide a more effective strategy for antibodydrug delivery into the CNS. Additionally, these in vivo resultsdemonstrate that a bispecific antibody can greatly improve the potencyof a promising anti-amyloidogenic therapeutic by significantlyincreasing brain penetrance of the targeting antibody drug. This couldbe highly advantageous, as enhanced drug delivery would translate toless potential side effects due to lower therapeutic dosing required.More generally, this technology has vast potential to be applied totherapeutics for a wide range of CNS diseases and represents an improvedapproach to provide safer antibody drugs.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1. A method of transporting a compound across the blood-brain barriercomprising exposing an antibody which binds with low affinity to ablood-brain barrier receptor (BBB-R) coupled to a compound to theblood-brain barrier such that the antibody transports the compoundcoupled thereto across the blood-brain barrier
 2. A method of increasingexposure of the CNS to a compound, wherein the compound is coupled to anantibody which binds with low affinity to a BBB-R, thereby increasingthe exposure of the CNS to the compound.
 3. A method of decreasingclearance of a compound administered to a subject, wherein the compoundis coupled to an antibody which binds with low affinity to a BBB-R, suchthat the clearance of the compound is decreased.
 4. A method ofincreasing retention in the CNS of a compound administered to a subject,wherein the compound is coupled to an antibody which binds with lowaffinity to a BBB-R, such that the retention in the CNS of the compoundis increased.
 5. A method of optimizing the pharmcokinetics and/orpharmacodynamics of a compound to be efficacious in the CNS in asubject, wherein the compound is coupled to an antibody which binds withlow affinity to a BBB-R, and the antibody is selected such that itsaffinity for the BBB-R after coupling to the compound results in anamount of transport of the antibody conjugated to the compound acrossthe BBB that optimizes the pharmacokinetics and/or pharmacodynamics ofthe compound in the CNS.
 6. A method of treating a neurological disorderin a mammal comprising treating the mammal with an antibody that binds aBBB-R and is coupled to a compound, wherein the antibody has beenselected to have a low affinity for the BBB-R and thereby improves CNSuptake of the antibody and coupled compound.
 7. The method of claim 1,wherein the compound is a neurological disorder drug or an imagingagent.
 8. The method of claim 2, wherein the increase is measuredrelative to a typical antibody not having lowered affinity for theBBB-R.
 9. The method of claim 1, wherein the antibody does not impairthe binding of the BBB-R to one or more of its native ligands.
 10. Themethod of claim 1, wherein the blood-brain barrier is in a mammal 11.The method of claim 10, wherein the mammal has a neurological disorder12. The method of claim 11, wherein the neurological disorder isselected from the group consisting of Alzheimer's disease (AD), stroke,dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophiclateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddlesyndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer,and traumatic brain injury
 13. The method of claim 10 wherein the mammalis a human.
 14. The method of claim 1, wherein the antibody has an IC50for the BBB-R from about 1 nM to about 100 μM.
 15. The method of claim8, wherein the IC50 is from about 5 nM to about 100 μM.
 16. The methodof claim 8, wherein the IC50 is from about 50 nM to about 100 μM. 17.The method of claim 8, wherein the IC50 is from about 100 nM to about100 μM.
 18. The method of claim 1, wherein the antibody has an affinityfor the BBB-R from about 5 nM to about 10 μM.
 19. The method of claim 1,wherein the antibody coupled to the compound has an affinity for theBBB-R from about 30 nM to about 1 μM.
 20. The method of claim 1, whereinthe BBB-R is selected from the group consisting of transferrin receptor(TfR), insulin receptor, insulin-like growth factor receptor (IGFreceptor), low density lipoprotein receptor-related protein 8 (LRP8),low density lipoprotein receptor-related protein 1 (LRP1), andheparin-binding epidermal growth factor-like growth factor (HB-EGF). 21.The method of claim 20, wherein the BBB-R is transferrin receptor 22.The method of claim 21, wherein the antibody does not inhibit thebinding of TfR to transferrin.
 23. The method of claim 1, wherein theantibody coupled to the compound is administered at a therapeutic dose.24. The method of claim 23, wherein the therapeutic dose isBBB-R-saturating.
 25. The method of claim 1, wherein the antibody is amultispecific antibody and the compound optionally forms one portion ofthe multispecific antibody
 26. The method of claim 25 wherein themultispecific antibody comprises a first antigen binding site whichbinds the BBB-R and a second antigen binding site which binds a brainantigen.
 27. The method of claim 26, wherein the brain antigen isselected from the group consisting of beta-secretase 1 (BACE1), Abeta,epidermal growth factor receptor (EGFR), human epidermal growth factorreceptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein,CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2(LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, deathreceptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophinreceptor (p75NTR), and caspase 6
 28. The method of claim 26, wherein themultispecific antibody binds both TfR and BACE1
 29. The method of claim26, wherein the multispecific antibody binds both TfR and Abeta.
 30. Amethod of making an antibody useful for transporting a compound acrossthe BBB comprising selecting an antibody specific for a blood-brainbarrier receptor (BBB-R) because it has a desirably low affinity for theBBB-R.
 31. The method of claim 30, wherein the affinity for the BBB-R isfrom about 5 nM to about 10 μM
 32. The method of claim 30, wherein theantibody has an IC50 of from about 1 nM to about 100 μM.
 33. The methodof claim 30 wherein the antibody is selected from a panel of antibodiesbased upon the affinity of the selected antibody
 34. The method of claim30 wherein the antibody is engineered to have the affinity
 35. Themethod of claim 30 comprising coupling the antibody with a therapeuticcompound.
 36. The method of claim 35 wherein the therapeutic compound isa neurological disorder drug.
 37. The method of claim 30 comprisingmaking a multispecific antibody which comprises a first antigen bindingsite which binds the BBB-R and a second antigen binding site which bindsa brain antigen.
 38. An antibody which binds to a BBB-R, wherein theaffinity of the antibody for the BBB-R is from about 5 nM to about 10μM.
 39. The antibody of claim 38 wherein the affinity is from about 20nM to about 1 μM
 40. The antibody of claim 38 wherein the BBB-R isselected from the group consisting of transferrin receptor (TfR),insulin receptor, insulin-like growth factor receptor (IGF receptor),low density lipoprotein receptor-related protein 8 (LRP8), low densitylipoprotein receptor-related protein 1 (LRP1), and heparin-bindingepidermal growth factor-like growth factor (HB-EGF).
 41. The antibody ofclaim 40 wherein the BBB-R is transferrin receptor (TfR).
 42. Theantibody of claim 40 wherein the BBB-R is insulin receptor
 43. Theantibody of claim 38 wherein the BBB-R is a human BBB-R.
 44. Theantibody of claim 38 which is coupled with a neurological disorder drug.45. The antibody of claim 44 which is a multispecific antibody whichcomprises a first antigen binding site which binds the BBB-R and asecond antigen binding site which binds a brain antigen.
 46. Theantibody of claim 45 wherein the brain antigen is selected from thegroup consisting of beta-secretase 1 (BACE1), Abeta, epidermal growthfactor receptor (EGFR), human epidermal growth factor receptor 2 (HER2),Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prionprotein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloidprecursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase6.
 47. The antibody of claim 46 wherein the brain antigen is BACE1 48.The antibody of claim 46 wherein the brain antigen is Abeta.
 49. Theantibody of claim 38 which is an antibody fragment with an antigenbinding region that binds the BBB-R.
 50. The antibody fragment of claim49 which is a Fab fragment.
 51. The multispecific antibody of claim 45which is a full-length antibody or an antibody fragment.
 52. (canceled)53. (canceled)
 54. (canceled)
 55. (canceled)